Segmented micro RNA mimetics

ABSTRACT

This invention relates generally to segmented oligonucleotides capable of modulating gene expression. Specifically, the instant invention relates to segmented microRNA (miRNA) oligonucleotides, including segmented miRNA precursors and segmented pre-microRNAs. The invention also relates to compositions comprising such segmented oligonucleotides, as well as to methods of making and using such oligonucleotides for diagnosis and treatment of diseases associated or causally linked to aberrant levels or activities of gene expression, including aberrant levels of coding and/or non-coding RNA.

PRIORITY APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/747,264, filed Jun. 23, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/391,703, filed Aug. 24, 2010, which is aNational Stage Entry of PCT Application No. PCT/US 10/46551, filed Aug.24, 2010 which claims the benefit of U.S. Patent Application No.61/236,486 filed Aug. 24, 2009.

TECHNICAL FIELD

This invention relates generally to segmented oligonucleotides capableof modulating gene expression. Specifically, the instant inventionrelates to segmented microRNA (miRNA) mimetic oligonucleotides,including segmented miRNA precursors and segmented pre-microRNAs. Theinvention also relates to compositions comprising such segmentedoligonucleotides, as well as to methods of making and using sucholigonucleotides for diagnosis and treatment of diseases associated orcausally linked to aberrant levels or activities of gene expression,including aberrant levels of coding and/or non-coding RNA.

BACKGROUND

Segmented oligonucleotides based on short interfering RNA (siRNA) havebeen evaluated for RNA interference (RNAi) activity. Leuschner et al.,(2006 EMBO 7:314) described an RNA-induced silencing complex which has adiscontinued passenger (or sense) strand and a 2′-O-methyl modifiednucleotide at position 9 of the passenger strand (5′ to 3′), the naturalcleavage site. Bramsen et al., (2007 Nucleic Acids Res. 35:5886)described an RNAi-active siRNA molecule comprising an internallysegmented passenger strand, where the nick or gap is not necessarilylocated at the natural cleavage site, stabilized with locked nucleicacid (LNA) modifications at a number of positions. See also, e.g.,Wengel et al., PCT Publication WO 2007/107162 A2; Quay et al., PCTPublication WO 2008/049078. None of Leuschner, Bramsen, Wengel and Quaydescribed RNAi-active molecules having discontinued guide (or antisense)strands. In fact, Bramsen and Wengel indicated that duplexes designed tocontain discontinuities in the guide strands completely eliminatedsilencing of the target.

The mechanistic difference between miRNA-mediated RNAi andsiRNA-mediated RNAi can make certain modifications and/or designssuitable for one but not the other. Thus, there remains a heightenedinterest in formulating new and advantageous design features suitablefor miRNA mimetics.

SUMMARY OF THE INVENTION

The specification describes certain segmented double-stranded miRNAmimetics having at least one non-contiguous (or discontinuous) strandcomprising a miRNA sequence, which can be introduced or applied tocells, tissues, and/or organisms to mediate RNAi. These molecules arereferred to herein as segmented miRNA mimetics, which comprise a guidestrand and a passenger strand. The guide strand comprises at least twocontiguous stretches of nucleotides separated by a discontinuity. Thepassenger strand can be fully contiguous, or alternatively can alsocomprise at least two contiguous stretches of nucleotides separated by adiscontinuity. Segmented miRNA mimetics of the invention therefore haveat least one non-contiguous guide strand comprising one or more miRNAsequences, or a portion thereof, including the seed sequence of suchmiRNA sequences. Non-limiting examples of miRNA sequences are thoseselected from the miRBase as of the filing date of the presentinvention; see for example Griffiths-Jones (2006) miRBase: the microRNAsequence database. Methods in Molecular Biology 342: 129-138 and miRBaserelease 13.0; http://micrirna.sanger.ac.uk/. A segmented miRNA of theinvention therefore can include one or more miRNA sequences selectedfrom SEQ ID NOs: 1-1090 of Table I, including portions thereof, such asthe seed sequences.

A segmented miRNA mimetic of the invention comprises at least onediscontinuity in the guide strand, and optionally at least onediscontinuity in the passenger strand that can be the same or differentas the discontinuity in the guide strand. Such discontinuities includenicks, gaps, substitutions, and/or insertions. Segmented miRNA mimeticcan comprise mixtures of different discontinuities in one or bothstrands.

A segmented miRNA mimetic of the invention comprises about 12 to about26 nucleotides in each strand, and further comprises about 10 to about26 base pairs between the strands. Thus, a prototypical segmented miRNAmimetic of the invention generally comprises two strands havingcomplementarity to form a duplex, each strand having between about 12 toabout 26 nucleotides, wherein the guide strand comprises any of SEQ IDNOs: 1-1090 or a portion thereof, and wherein the guide strand furthercomprises at least on discontinuity.

Segmented miRNA mimetics of the invention can be administered to a cell,a tissue or an organism to supplement or increase the levels of theircorresponding endogenous miRNAs and hence potentiate RNAi activityagainst their corresponding miRNAs targets. Because each endogenousmiRNA typically has multiple targets, an exogenously introducedsegmented miRNA mimetic of the invention does not necessarily share thesame number, identity or type of targets with its correspondingendogenous miRNA. However, the exogenously-introduced segmented miRNAmimetic exerts activity on at least one (i.e., one or more or all) ofthe targets of its corresponding endogenous miRNA.

A segmented miRNA mimetic can be chemically modified at the nucleic acidbase, phosphodiester backbone, or sugar to achieve, for instance,increased stability and/or reduced immunogenicity, and otherpharmaceutically desirable attributes, including properties that wouldallow for enhanced delivery or lower toxicity. Methods of chemicallymodifying oligonucleotides to achieve such ends are known in the art.For instance, numerous such methods are set forth in McSwiggen, et al.,U.S. Publication No. 2006/0211642.

In a further aspect, the specification provides a composition comprisingone or more (i.e., in the number of individual molecules and/or intypes) segmented miRNA mimetics in a pharmaceutically acceptable carrieror diluent. In another aspect, the specification provides a method ofintroducing or applying one or more segmented miRNA mimetics to cells(regardless of whether the RNAi or other gene modulation process takesplace inside the cells, outside the cells, or on the cell-membrane),tissues, organisms, or reconstituted in vitro systems, to increase thelevels of corresponding endogenous miRNAs. Embodiments of the inventioninclude methods of modulating gene expression, biologic pathways, orphysiologic pathways in cells, cultures, tissues, or organisms such assubjects or patients, comprising administering one or more segmentedmiRNA mimetics of the invention in an amount that is sufficient tomodulate the expression of one or more genes that are regulated by thecorresponding endogenous miRNAs. In a specific embodiment, more than onetype of segmented miRNA mimetic is administered. For example, a numberof different segmented miRNA mimetics of the invention can beadministered concurrently, in sequence, or in an ordered progression.

In certain embodiments, administration of the composition(s) can beenteral or parenteral. In certain aspects, enteral administration isoral. In further aspects, parenteral administration is intralesional,intravascular, intracranial, intrapleural, intratumoral,intraperitoneal, intramuscular, intralymphatic, intraglandular,subcutaneous, topical, intracronchial, intratracheal, intranasal,inhaled, or instilled. Compositions of the invention can be administeredregionally or locally, and not necessarily directed into a lesion.

Embodiments of the invention can include obtaining or assessing a geneexpression profile or miRNA profile of a target cell, tissue, ororganism prior to selecting the mode of treatment, by, for example,administration of one or more segmented miRNA mimetics. In certainaspects of the invention, one or more segmented miRNA mimetics canmodulate a single gene. In a further aspect, one or more genes in one ormore genetic, cellular, or biologic/physiologic pathways can bemodulated by a single segmented miRNA mimetic or a complement thereof,alone or in combination with other miRNAs or mimetics, or with othernucleic acid-based gene modulators, such as siRNAs, antisense molecules,ribozyme molecules, and the like.

A further aspect of the invention is directed to a method of modulatinga cellular pathway comprising administering to the cell an amount of asegmented miRNA mimetic, alone or in combination with other miRNAs,mimetics, siRNAs, or other suitable nucleic-acid based or non-nucleicacid based agents capable of modulating one or more relevant genes inthe same or associated pathways. In a related aspect, the invention isdirected to methods of modulating a cellular pathway comprisingadministering to the cell a segmented miRNA mimetic in an amountsufficient to modulate the gene expression, function, status, or stateof a cellular pathway, in particular a pathway that is known to includeone or more genes associated with the corresponding endogenous miRNA.Modulation of a cellular pathway includes, but is not limited to,modulating the expression of one or more genes associated with thepathway. Modulation of a gene includes inhibiting its function, alsocalled “down-regulate a gene,” or providing an agonist to augment itsfunctional, also called “up-regulating a gene.” What is modulated iseither the expression level or activity of a gene or its related geneproduct or protein.

Compositions and methods comprising a segmented miRNA mimetic are alsouseful for treating diseases or disorders associated with aberrantexpression levels or activity of one or more corresponding miRNAtargets. These diseases and/or disorders include, for example,hyperproliferative disorders (e.g., cancer), inflammatory conditions(e.g., arthritis), respiratory diseases, pulmonary diseases,cardiovascular diseases, autoimmune diseases, allergic disorders,neurologic diseases, infectious diseases (e.g., viral infections), renaldiseases, transplant rejections, or any other conditions that respond tosuch modulation.

Still a further embodiment includes methods of treating a patient with apathological condition comprising one or more steps: (a) administeringto the patient an isolated or a synthetic segmented miRNA mimetic of theinvention in an amount sufficient to modulate the expression of acellular pathway; and (b) administering a second therapy, wherein themodulation of the cellular pathway in (a) sensitizes the patient to thesecond therapy. A cellular pathway can include, but is not limited to,one or more pathways that are known to be associated with known miRNAslisted in the miRBase as of the date of filing of the instantapplication. A second therapy can include administration of one or moremiRNAs or mimetics targeting the same or different mRNAs, or one or moreother therapeutic nucleic acids. A second therapy can also be oneselected from other standard therapies, such as chemotherapy, radiationtherapy, drug therapy, immunotherapy, and the like.

The invention also features a kit or article of manufacture comprisingone or more segmented miRNA mimetics, typically in a pharmaceuticalcomposition, and instructions for administering the composition to treata pathological condition. Optionally, the kit or article of manufacturecan contain one or more other pharmaceutical compositions or agents andinstructions for their use in conjunction with the pharmaceuticalcomposition comprising the segmented miRNA mimetics.

In yet a further aspect of the invention, one or more segmented miRNAmimetics of the invention can be included in a kit or article ofmanufacture for assessment or diagnosing of a pathological condition orthe risk of developing a pathological condition.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein and that different embodiments can be combined.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C, 2A-2B, 3, 4A-4B, 5 and 6 illustrate examples of differentstructural features of segmented miRNA mimetics. It will be apparent tothe skilled person in the art that the various structural featuresillustrated in these figures can be combined. For example, a particularpattern of the overhangs can be combined with a molecule comprising oneor more discontinuities at particular positions, which can or can not belinked, by phosphodiester bonds or non-phosphodiester connectors.Therefore, these figures should be interpreted as merely representativebut non-limiting, such that, for example, the nature and position of thediscontinuities can be changed and additional features, such as, forexample, overhangs and nucleotide analogs, can be introduced to the samemolecules.

FIGS. 1A, 1B, and 1C illustrate the basic structural features of asegmented miRNA mimetic. FIG. 1A shows 3 representative segmented miRNAmimetics, comprising nicks or gaps in the guide (the lower strand), orin both the passenger (upper strand) and the guide strands. Each boxrepresents a nucleotide (including a nucleotide analog), or anon-nucleotide substitute moiety. Each line connecting the boxesrepresents a phosphodiester bond or non-phosphodiester connector.Distinct contiguous stretches of nucleotides in a given strand arecolored/shaded differently to facilitate the identification of the nickor gap positions. FIG. 1B shows a representative conformation of asegmented miRNA mimetic, wherein the passenger strand is uninterruptedand wherein the guide strand comprises a gap of 1 nucleotide. A similarmotif comprising a nick instead of a gap is contemplated by the instantinvention. FIG. 1C shows a representative segmented miRNA mimeticwherein each of the passenger strand and the guide strand comprises anick. Some of the discontinuities in this figure are shown as nicks.However, those skilled in the art would appreciate that gaps can bepresent at similar positions, and other types of discontinuities such assubstitutions and insertions are also contemplated. Moreover, thoseskilled in the art would appreciate that a gap can be a stretch of 1 toabout 10 contiguous vacant nucleotide positions.

FIG. 2A illustrates various overhangs and blunt ends can be utilized inthe design of a segmented miRNA mimetic of the invention. Thediscontinuities are shown in this figure as nicks or gaps of a singlenucleotide, but other kinds of discontinuities, including larger gaps ofone or more (e.g., up to 10) nucleotides, as well as substitutionsand/or insertions are also contemplated.

FIG. 2B illustrates a representative segmented miRNA mimetic wherein oneset of the terminal ends are connected by a linker.

FIG. 3 illustrates that one or more discontinuities (nicks or gaps areshown, but also substitutions and insertions) can be present in theguide strand, or in both the passenger and the guide strands.

FIGS. 4A and 4B illustrate that the sizes or positions of thediscontinuities on the guide strand, or both the passenger and guidestrands can vary. The molecules are named according to the starting andending positions of the discontinuities from the 5′-end.

FIG. 5 illustrates that one or more suitable non-nucleotidesubstitutions or insertions can be used to connect a pair of neighboringcontiguous stretches of nucleotides in the passenger strand, the guidestrand, or both the passenger and guide strands.

FIG. 6 illustrates certain segmented miRNA mimetics comprising invertedabasic modifications at the internal ends. The molecules are namedaccording to the starting and ending positions of the discontinuities.

FIGS. 7, 8, 9A-9B, 10A-10B, 11A-11B, 12A-12B, 13A-13B, 14A-14D, 15A-15B,16, 17A-17B, 18A-18B, 19A-19B and 20A-20B show experimental dataobtained from the examples herein.

FIG. 7 illustrates RNAi activity against an endogenous miR-124 target,CD164. RNAi activity of various segmented miRNA mimetics derived frommiR-124 (“segmented miR-124”) was measured. Levels of knockdown achievedby the segmented miR-124 constructs and by the correspondingnon-segmented miR-124 mimetic, comprising the endogenous mature miR-124sequence as its guide strand, were determined. The segmented miR-124constructs can comprise one or more locked nucleic acid (“LNA”)nucleotides in one or both strands, each represented by an underlinednucleotide in Table III herein, although the LNAs are not necessarilyplaced at the underlined nucleotide positions.

FIG. 8 illustrates RNAi activity against another endogenous miR-124target, VAMP3. RNAi activity of various segmented miR-124 mimetics wasmeasured. Levels of knockdown achieved by the segmented miR-124constructs and by the corresponding non-segmented miR-124 mimetic,comprising the endogenous mature miR-124 sequence as its guide strand,were determined. The segmented miR-124 constructs can comprise one ormore LNA nucleotides in one or both strands, each represented by anunderlined nucleotide in Table IV herein, although the LNAs are notnecessarily placed at the underlined nucleotide positions.

FIG. 9A illustrates RNAi activity of various segmented miR-124 mimetics(i.e., of Table V) against CD164. Levels of knockdown achieved by thesegmented miR-124 constructs and by the corresponding non-segmentedmiR-124 mimetic, comprising the endogenous mature miR-124 sequence asits guide strand, were determined. Knockdown or inhibition, or the lackthereof, by segmented miR-34 constructs, which were designed based onhuman miR-34, is presented as a negative control.

FIG. 9B illustrates RNAi activity against an endogenous miR-34 target,TK1, of various segmented miRNA mimetics derived from miR-34 (“segmentedmiR-34”) (i.e., molecules of Table V). Levels of knockdown achieved bythe segmented miR-34 constructs and by the corresponding non-segmentedmiR-34 mimetic, comprising the endogenous mature miR-34 sequence as itsguide strand, were determined. Knockdown or inhibition, or the lackthereof, by segmented miR-124 constructs, is presented as a negativecontrol.

FIG. 10A illustrates RNAi-mediated activity against CD164 by segmentedmiR-124 constructs comprising one or more inverted abasic modifiedinternal ends (i.e., molecules of Table VI). FIG. 10B illustratesRNAi-mediated activity against VAMP3 by segmented miR-124 constructscomprising one or more inverted abasic modified internal ends.

FIG. 11A illustrates RNAi-mediated activity against CD164 by segmentedmiRNA-124 constructs comprising abasic substitutions (i.e., molecules ofTable VII). FIG. 11B illustrates RNAi-mediated activity against VAMP3 bysegmented miR-124 constructs comprising abasic substitutions.

FIGS. 12A and 12B demonstrate that miR-124 activity toleratessegmentation of the passenger or guide strands. FIG. 12A includesschematic illustrations of miR-124 duplex designs, wherein the topstrand represents the passenger strand and the bottom strand representsthe guide strand. The gray-shade change indicates the site of a break inthe strand backbone. The dark gray circle in the G10Cy3.12/P schematicrepresents a 5′-Cy3 label, and the light gray circle in the G10i.12/Pschematic indicates a 5′ inverted abasic nucleotide. FIG. 12Billustrates the dose-dependent response of CD164, a known miR-124target, to various concentrations of the designs shown in FIG. 12A inHCT-116 cells as measured by RT-qPCR. UC3 corresponds to the negativecontrol oligomer. EC50s for each curve are as follows: G/P, 0.12 nM;G/P10.12, 0.29 nm; G10.12/P, 0.22 nM; G10.11/P, 0.53 nM; G10i.12/P, 0.21nM: G10Cy3.12/P, 0.45 nm.

FIGS. 13A and 13B provide a microarray analysis showing seed-basedactivity from a segmented guide strand. FIG. 13A is a graphicillustration of the microarray signature 24 hours after transfection of10 nM G10.12/P, plotted as expression ratio (relative to mocktransfection) versus fluorescence intensity. Significantly downregulated probes (P<1 e-6) are seen as gray data points below the blackdata points and unregulated probes are seen as gray data points abovethe black data points. Hypergeometric analysis of the hexamer content ofthe down regulated UTRs showed that the most significantly enrichedhexamer (P<1e-20) was GCCTTA, corresponding to positions 2-7 of thetransfected miR-124. FIG. 13B provides a comparison of gene expressiondata from G10.12/P transfected cells and G/P transfected cells.Expression ratio of the G/P transfection versus the mock transfection isplotted on the x-axis and the G10.12/P expression ratio versus mocktransfection on the y-axis. The weighted correlation coefficient wascalculated as 0.9, illustrating the similar effects of both RNAcomplexes on gene expression.

FIGS. 14A, 14B, 14C, and 14D illustrate that the activity of anunsegmented miR-124 guide strand is enhanced by additionalcomplementarity beyond the seed sequence, but activity of a segmentedguide strand is not. FIG. 14A is a schematic of miR-124 target sitesthat were duplicated and inserted into dual luciferase reporter vectors.FIG. 14B illustrates that G/P miR-124 exhibits suppression of activityfrom a reporter with a seed sequence match, which is enhanced byreporters containing additional complementarity to the 3′ end of theguide strand (2×7a3p) or full-length complementarity. The EC50s for thecurves: 2×7a, 0.39 nM; 2×7a3p, 0.08 nM; 2×FL, 0.09 nM. The EC50s for thecurves are: 2×7a, 0.41 nM; 2×7a3p, 0.06 nM; 2×FL, 0.09 nM. FIG. 14Cillustrates that segmentation of the passenger strand preserves thetrends seen in FIG. 14B. FIG. 14D shows that activity from reporterswith 3′ complementarity is identical to that observed from the seed-onlyreporter when the guide strand is segmented. The EC50s for the curvesare: 2×7a, 0.89 nM; 2×7a3p, 0.61 nM; 2×FL, 0.80 nM.

FIGS. 15A and 15B illustrate RNAi activity mediated by a segmentedmiR-124 precursor. A 58-mer miR-124 precursor was designed andtransfected into HCT-116 cells at varying concentrations. FIG. 15A is amiR-124 precursor schematic. FIG. 15B is a graph showing CD164 knockdownas measured by RT-qPCR. Activity of the unsegmented precursor iscomparable to that of the mature miRNA, while activity of the segmentedprecursor is reduced but is significantly above background. The EC50sfor the curves are: G/P, 0.14 nM; hairpin precursor, 0.31 nM; segmentedprecursor, 1.48 nM.

FIG. 16 illustrates that a segmented guide strand exhibits decreasedknockdown for targets containing 3′ supplementary pairing. Microarraydata shown in FIG. 13B was further analyzed, specifically to analyze1057 downregulated genes that contain TargetScan seed matches. TheTargetScan 3′ pairing score was then calculated for these genes, and thedownregulation of the top quartile of 3′ scores (containing a highdegree of 3′ pairing) was compared with the downregulation of the bottomquartile of 3′ scores. The cumulative distribution of the difference indownregulation (between G/P and G10.10/P) is plotted for the top andbottom quartiles. A Kolmogorov-Smirnov test (p=0.02) shows that the topquartile of genes shows less knockdown in G10.10/P versus G/P, ascompared with the bottom quartile.

FIG. 17A illustrates knockdown of CD164 expression by segmented miR-124mimetics comprising deletions and c3 or c6 substitutions, while FIG. 17Billustrates knockdown of VAMP3 expression by these segmented microRNAs.

FIG. 18A illustrates knockdown of CD164 expression by segmented miR-124comprising c3 substitutions, while FIG. 18B illustrates knockdown ofVAMP3 expression by these segmented microRNAs.

FIG. 19A illustrates knockdown of CD164 expression by segmented miR-124comprising c6 substitutions, while FIG. 19B illustrates knockdown ofVAMP3 expression by these segmented microRNAs.

FIG. 20A illustrates knockdown of CD164 expression by segmented miR-124comprising c3 and c6 insertions, while FIG. 20B illustrates knockdown ofVAMP3 expression by these segmented microRNAs.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

“About” as used herein indicates that a value includes the standarddeviation of error for the device or method being employed to determinethe value.

“Analog” as used herein refers to its meaning as is generally acceptedin the art. The term generally refers to a compound that is structurallysimilar to a parent compound (e.g., a nucleotide), but differs incomposition (e.g., one or more atom(s) or functional group(s) is/aredifferent, added, or removed). The analog can have different chemical orphysical properties than the original parent compound and can haveimproved biological or chemical activity. For example, the analog can bemore hydrophilic or it can have altered activity of the parent compound.The analog can be a naturally or non-naturally occurring (e.g.,chemically-modified or recombinant) variant of the original parentcompound. An example of an RNA analog is an RNA molecule comprising anucleotide analog. An example of a nucleotide analog is a nucleotidethat is chemically modified at the sugar, base or nucleoside, as isgenerally known in the art.

The term “aptamer” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to a nucleic acidmolecule that binds specifically to a target molecule wherein thenucleic acid molecule has sequence that comprises a sequence recognizedby the target molecule in its natural setting. Alternately, an aptamercan be a nucleic acid molecule that binds to a target molecule whereinthe target molecule does not naturally bind to a nucleic acid. Thetarget molecule can be any molecule of interest. For example, theaptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art (see, e.g., Gold et al, 1995 Annu.Rev. Biochem. 64:163; Brody and Gold, 2000 J. Biotechnol. 74:5; Sun,2000 Curr. Opin. Mol. Ther. 2: 100; Kusser, J. 2000 Biotechnol. 74:21;Hermann and Patel, 2000 Science 257:820; and Jayasena, 1999 ClinicalChem. 45:1628).

As described herein, a “base pair” can be formed between twonucleotides, a nucleotide and a modified nucleotide, two modifiednucleotides, a nucleotide and a nucleotide analog, two nucleotideanalogs, a nucleotide and a non-nucleotide substitute moiety, or twonon-nucleotide substitute moieties. In a specific embodiment, anon-nucleotide substitute can comprise any chemical moiety that iscapable of associating with a component of the cellular RNAi machinery,such as, for example, the PAZ domain, the PIWI domain, and/or otherArgonaute protein domains associated with the RISC. Non-traditionalWatson-Crick base pairs are also understood as “non-canonical basepairs,” which is meant any non-Watson Crick base pair, such asmismatches and/or wobble base pairs, including flipped mismatches,single hydrogen bond mismatches, trans-type mismatches, triple baseinteractions, and quadruple base interactions. Non-limiting examples ofsuch non-canonical base pairs include, but are not limited to, ACreverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric,CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-iminosymmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, ACamino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AUN1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GAamino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GCcarbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GGcarbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GUimino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3, GAcarbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H. AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

The term “biodegradable” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to degradationin a biological system, for example enzymatic degradation or chemicaldegradation.

The term “biodegradable linker” as used herein refers to its meaning asis generally accepted in the art. The term generally refers to a nucleicacid or non-nucleic acid linker molecule that is designed as abiodegradable linker to connect one molecule to another molecule, forexample, connecting a biologically active molecule to a segmented miRNAmimetic of the invention or to either the passenger and/or guide strandsof a segmented miRNA mimetic of the invention. The biodegradable linkercan be attached to a segmented miRNA mimetic of the invention at one ormore of the terminal ends, internal ends, or any other nucleotidepositions that is not vacant. The biodegradable linker is designed suchthat its stability can be modulated for a particular purpose, such asdelivery to a particular tissue or cell type. The stability of a nucleicacid-based biodegradable linker molecule can be modulated by usingvarious chemistries, for example combinations of ribonucleotides,deoxyribonucleotides, and chemically modified nucleotides, such as2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl,and other 2′-modified or base modified nucleotides. The biodegradablenucleic acid linker molecule can be a dimer, trimer, tetramer or longernucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotidesin length, or can comprise a single nucleotide with a phosphorus-basedlinkage, for example, a phosphoramidate or phosphodiester linkage. Thebiodegradable nucleic acid linker molecule can also comprise nucleicacid backbone, nucleic acid sugar, or nucleic acid base modifications.

The term “biologically active molecule” as used herein refers to itsmeaning as is generally accepted in the art. The term generally refersto compounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive molecules either alone or in combination with other moleculescontemplated by the instant invention include therapeutically activemolecules such as antibodies, hormones, antivirals, peptides, proteins,chemotherapeutics, small molecules, vitamins, co-factors, nucleosides,nucleotides, oligonucleotides, enzymatic nucleic acids, guide nucleicacids, triplex forming oligonucleotides, 2,5-A chimeras, siRNA, miRNAmimetics, dsRNA, allozymes, aptamers, decoys and analogs thereof.Biologically active molecules of the invention also include moleculescapable of modulating the pharmacokinetics and/or pharmacodynamics ofother biologically active molecules, for example, lipids and polymerssuch as polyamines, polyamides, polyethylene glycol and otherpolyethers.

By “capable of participating in RNAi against endogenous RNA targets oftheir corresponding naturally-occurring miRNAs” is meant that, when RNAiactivity is measured by a suitable in vivo or in vitro assay or method,a segmented miRNA mimetic molecule of the invention demonstrates atleast 5% or more of the knockdown effect against a target of itscorresponding naturally-occurring miRNA as compared to the knockdowneffect achieved by a non-segmented miRNA mimetic molecule directed tothe same target under same experimental conditions. Preferably, asegmented miRNA mimetic molecule of the invention is capable ofachieving 25% or more, 35% or more, 50% or more, 55% or more, 60% ormore, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more,90% or more, 95% or more, 99% or more, or even 100% or more (i.e., equalor more potent RNAi activity) knockdown of the target than anon-segmented miRNA mimetic against the same target.

The term “cap structure” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to chemicalmodifications, which have been incorporated into the ends ofoligonucleotide (see, for example, Matulic-Adamic et al., U.S. Pat. No.5,998,203, incorporated by reference herein). These terminalmodifications can protect certain nucleic acid molecules fromexonuclease degradation, and can impart certain advantages in deliveryand/or cellular localization.

As used herein, the term “complementary” (or “complementarity”) refersto its meaning as is generally accepted in the art. The term generallyrefers to nucleic acid sequences that are capable of base-pairingaccording to the standard Watson-Crick complementarity rules, that ispurines will base pair with pyrimidines to form combinations: guaninepaired with cytosine (G:C); and adenine paired with either thymine (A:T)in the case of DNA, or adenine paired with uracil (A:U) in the case ofRNA. Base-pairing according to the Standard Waston-Crick complementarityrules can include base pairs formed between modified or nucleotideanalogs. Aside from forming hydrogen bond(s) with each other accordingto the traditional Waston-Crick rules, a nucleic acid sequence can formother non-traditional types of base pairing with another nucleic acidsequence, and as such, the two nucleic acid sequences can also be called“complementary.” As used herein, the term “complementary” thusencompasses any “base-pairing,” which can be by hydrogen bonds or by anyother interactions, of nucleotides, modified nucleotides, analogs,and/or non-nucleotides that provide sufficient binding free energybetween the strands to allow the relevant function of the segmentedmiRNA mimetic, e.g., RNAi activity, to proceed. Determination of bindingfree energies for nucleic acid molecules is known in the art (see, e.g.,Turner et al., 1987 CSH Symp. Quant. Biol. LII:123; Frier et al., 1986Proc. Nat. Acad. Sci. USA 83:9373; Turner et al., 1987 J. Am. Chem. Soc.109:3783).

A percent complementarity indicates the percentage of contiguousresidues in a first nucleic acid molecule that can form hydrogen bonds(e.g., in Watson-Crick base-pairing) with a second nucleic acidsequence. For example, a first nucleic acid molecule can have 10nucleotides and a second nucleic acid molecule can have 10 nucleotides,then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the firstand second nucleic acid molecules, which can or can not form acontiguous double-stranded region, represents 50%, 60%, 70%, 80%, 90%,or 100% complementarity, respectively. Complementarity can be foundbetween two regions of a same nucleic acid molecule, such as, forexample, in a hairpin loop or a stem loop structure. In otherembodiments, complementarity can be found between two different nucleicacid molecules, such as, for example, in a segmented miRNA mimetic ofthe invention comprising distinct and separate passenger and guidestrands.

In keeping with the usual practice by those of ordinary skill in theart, when the passenger strand and guide strand of the correspondingnon-segmented miRNA are aligned on paper, (with the passenger strandarranged from 5′ to 3′ (left to right) and the guide strand arrangedfrom 3′ to 5′ (left to right)) such that the each pair of complementary(base-pairing) nucleobases are located at directly opposite positions inthe passenger and guide strand, the relative positions of thebase-pairing nucleotides are termed “complementary nucleotidepositions.” It is often helpful to mark the position of the nucleotidesin the non-segmented miRNA mimetic and use those positions to marknicks, gaps, substitutions, or insertions introduced into acorresponding segmented mimetic construct. Typically the firstnucleotide position at the 5′-end of the passenger strand of anon-segmented duplex miRNA mimetic is position 1 of passenger strand,the nucleotide immediately adjacent to it is position 2, and so on andso forth. Likewise, the first nucleotide position at the 5′-end of theguide strand of the non-segmented duplex miRNA mimetic is position 1 ofthe guide strand, the nucleotide immediately adjacent to it is position2, and so on and so forth.

By “a contiguous stretch of nucleotides” or “a contiguous stretch ofnucleotide positions” is meant a continuous series of at least 2nucleotides or at least two nucleotide positions. For example, acontiguous stretch of nucleotides can refer to an unsegmented oruninterrupted oligonucleotide of 2 to 20 nucleotides in length. Whenreferring to a contiguous stretch of nucleotides, the bonds connectingthe nucleotides within the stretch can be phosphodiester bonds ornon-phosphodiester linkages. A gap comprising a contiguous stretch ofnucleotide positions can refer to a gap occupying, for example, from 1to 10 or more nucleotide positions.

A segmented miRNA mimetic of the invention provided to a cell istypically designed based on the sequence of a naturally-occurring miRNAin the cell. As such, the naturally-occurring miRNA in the cell isreferred to herein as “the corresponding miRNA.” A segmented miRNAmimetic of the invention provided to a cell is also understood to targetone or more target mRNAs that are also targeted by the correspondingnaturally-occurring miRNA. As such, each RNA targeted by thecorresponding naturally-occurring miRNA is referred to as “thecorresponding miRNA target.” It is contemplated that a segmented miRNAmolecule introduced to a cell is not necessarily or does not necessarilycomprise a nucleic acid sequence that is identical, essentiallyhomologous, or even substantially homologous to a naturally-occurringmiRNA, but the segmented miRNA is capable of either becoming orfunctioning as a naturally-occurring miRNA under appropriate conditions.

The term “discontinuity” as used herein refers to a non-contiguoussegment of the nucleotide sequence of the guide strand, passenger strandor both the passenger and guide strands of a segmented micro RNAmimetic. A discontinuity can include one or more nicks, gaps,substitutions or insertions. The discontinuity can comprise, forexample, from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) ormore unoccupied or vacant nucleotide positions in the guide strand, thepassenger strand, or both the guide and passenger strands. For example,a nick will comprise 0 unoccupied or vacant nucleotide positions,whereas a gap will comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 910 or more) vacant or unoccupied nucleotide positions. Likewise, thediscontinuity can comprise, for example, from 0 to 10 (e.g., 0, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10) or more nucleotide positions that are occupiedor replaced by a non-nucleotide moiety in the guide strand, thepassenger strand, or both the guide and passenger strands. For example,an insertion will comprise a non-nucleotide moiety that can occupy 0nucleotide positions, whereas a substitution will comprise anon-nucleotide moiety that occupies one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9 10 or more) otherwise vacant nucleotide positions.

As used herein, “endogenous” refers to its meaning as is generallyaccepted in the art. The term generally refers to any material from orproduced inside an organism, cell, tissue or system. As used herein, an“endogenous miRNA” is a naturally-occurring miRNA in a cell, tissue,organism, including a mammal, such as, for example, a human. “Exogenous”generally refers to any material introduced from or produced outside anorganism, cell, tissue or system.

The term “expression” refers to its meaning as is generally accepted inthe art. The term generally refers to the transcription and/ortranslation of a particular nucleotide sequence, for example when drivenby its promoter.

The term “gap” as used herein refers to a contiguous stretch of one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more), internal (asopposed to “terminal”) vacant, unoccupied or “unfilled” nucleotidepositions in one or both strands of a segmented miRNA mimetic of theinvention. The gap can be present in the guide strand, the passengerstrand, or in both guide and passenger strands of a segmented microRNAmimetic of the invention.

The term “gene” as used herein, especially in the context of “targetgene” for an RNAi agent, refers to the meaning as is generally acceptedin the art. The term generally refers to a nucleic acid (e.g., “targetDNA” or “target RNA”) sequence that comprises partial length or entirelength coding sequences necessary for the production of a polypeptide.The target gene can also include a UTR (i.e., untranslated region) ornon-coding region of the nucleic acid sequence. A gene or target genecan also encode a functional RNA (fRNA) or non-coding RNA (ncRNA) as aregenerally known in the art, such as endogenous antisense RNA, smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof, or any otherregulatory RNA or precursor thereof. Such non-coding RNAs can serve astarget nucleic acid molecules for RNA interference in modulating theactivity of fRNA or ncRNA involved in functional or regulatory cellularprocesses. Aberrant fRNA or ncRNA activity leading to disease cantherefore be modulated by the RNAi agents of the invention. RNAi agentstargeting fRNA and ncRNA can also be used to manipulate or alter thegenotype or phenotype of a subject, organism or cell, by intervening incellular processes such as genetic imprinting, transcription,translation, or nucleic acid processing (e.g., transamination,methylation etc.). A target gene can be a gene derived from a cell, anendogenous gene, a transgene, or exogenous genes such as genes of apathogen, for example a virus, which is present in the cell afterinfection thereof. A cell containing a target gene can be derived fromor contained in any organism, for example a plant, animal, protozoan,virus, bacterium, or fungus. Non-limiting examples of plants includemonocots, dicots, or gymnosperms. Non-limiting examples of animalsinclude vertebrates or invertebrates. Non-limiting examples of fungiinclude molds or yeasts. For a review, see for example Snyder andGerstein, 2003, Science 300:258-260. In one aspect of the presentinvention, a segmented miRNA mimetic is capable of exerting regulatoryeffects on multiple target genes. Also, at least one of these targetgenes, but typically more than one target genes, can be shared between asegmented miRNA mimetic of the invention and its correspondingendogenous miRNA.

As used herein, “gene silencing” refers to a partial or completeloss-of-function through targeted inhibition of an endogenous miRNAtarget in a cell. As such, the term is used interchangeably with RNAi,“knockdown,” “inhibition.” “down-regulation,” or “reduction” ofexpression of a miRNA target gene. Depending on the circumstances andbiological problem to be addressed, it can sometimes be preferable toincrease expression of one or more related genes, which is termed“up-regulation” herein. Alternatively, it might be desirable to reduceor increase gene expression as much as possible or only to a certainextent.

By “guide strand” of a segmented miRNA of the invention is meant two ormore distinct contiguous stretches of nucleotides at least one of whichis substantially homologous or identical to the whole or a part of asequence of a corresponding naturally-occurring miRNA, such as oneselected from the miRBase, and for example, such as one selected fromSEQ ID NOs: 1-1090 of Table I herein. The nucleotides within eachcontiguous stretch can be connected by traditional phosphodiester bondsand/or non-phosphodiester connectors. In addition, the guide strand of asegmented miRNA mimetic can comprise two or more distinct stretches ofnucleotides that are capable of forming base pairs with the nucleotidesor residues at the complementary nucleotide positions of the passengerstrand.

As used herein, the term “homologous” (or “homology”) refers to itsmeaning as is generally accepted in the art. The term generally refersto the number of nucleotides of the subject nucleic acid sequence thathas been matched to identical nucleotides of a reference nucleic acidsequence, typically by a sequence analysis program or by visualinspection. For example, nucleic acid sequences can be compared usingcomputer programs that align the similar sequences of nucleic acids andtherefore define the differences. Exemplary computer programs includesthe BLAST program (NCBI) and parameters used therein, as well as theDNAstar system (Madison, Wis.), which can be used to align sequencefragments. Equivalent alignments and assessments can also be obtainedthrough the use of any standard alignment software.

As used herein, the terms “including” (and any form thereof, such as“includes” and “include), “comprising” (and any form thereof, such as“comprise” and “comprises”), “having” (and any form thereof, such as“has” or “have”), or “containing” (and any form thereof, such as“contains” or “contain”) are inclusive and open-ended and do not excludeadditional, un-recited elements or method steps.

The term “insertion” as used herein refers to a discontinuity whereinone or more non-nucleotide moieties are incorporated into the guidestrand and/or passenger strand, while preserving the base pairs in theguide and passenger strands. Examples of such non-nucleotide moietiesare provided herein and others are provided as is generally known tothose of skill in the art.

The term “internal ends” refers to the ultimate nucleotides of thecontiguous stretches of nucleotides on either side of a gap or a nick.Gaps or nicks do not have “terminal ends” for the purpose of thisdisclosure.

As used herein, the term “internally unpaired nucleotides” refers tonucleotides, which do not form base pairs with nucleotides at thecomplementary nucleotide positions in the opposite strand according tothe standard Waston-Crick base-pairing rules. The term “internallyunpaired nucleotides” also refers to nucleotide analogs ornon-nucleotide residues that do not form hydrogen bonds or base pairswith the nucleotides, nucleotide analogs, or non-nucleotide residues atthe complementary nucleotide positions in the opposite strand.

In certain embodiments, a segmented miRNA of the invention can beisolated. As used herein, an “isolated” oligonucleotide is nucleic acidmolecule that exists in a physical form differing from any nucleic acidmolecules of identical sequence as found in nature. “Isolated” does notrequire, although it does not prohibit, that the nucleic acid bephysically removed from its native environment. For example, a nucleicacid can be said to be “isolated” when it includes nucleotides and/orinternucleotide bonds not found in nature. A nucleic acid can be said tobe “isolated” when it exists at a purity not found in nature, wherepurity can be adjudged with respect to the presence of nucleic acids ofother sequences, with respect to the presence of proteins, with respectto the presence of lipids, or with respect to the presence of any othercomponent of a biological cell, or when the nucleic acid lacks sequencethat flanks an otherwise identical sequence in an organism's genome, orwhen the nucleic acid possesses sequence not identically present innature. In aspects of the invention, a segmented miRNA is isolated byvirtue of its having been synthesized in vitro. It will be understood,however, that isolated nucleic acids can be subsequently mixed or pooledtogether.

As used herein, the term “locked nucleic acid” (LNA) refers to itsmeaning as is generally accepted in the art. The term generally refersto a structure of the general Formula I:

where X and Y are independently selected from the group consisting of—O—, —S—, —N(H)—, —N(R)—, —CH₂—, or —CH— (if part of a double bond),—CH₂—O—, CH₂—S—, CH₂—N(H)—, —CH₂—N(R)—, —CH₂—CH₂—, and CH₂—CH— (if partof a double bond), —CH═CH—, where R is selected from hydrogen andC₁₋₄-alkyl; Z and Z* are independently selected from an internucleotidelinkage, a terminal group or a protecting group; B constitutes a naturalor non-natural nucleobase; and the asymmetric groups can be found ineither orientation.

The 4 chiral centers of Formula I, as shown, are in a fixedconfiguration. But their configurations are not necessary fixed. Alsocomprised in the invention are compounds of the generally Formula I,wherein the chiral centers are found in different configurations, suchas those represented in Formula II (below). Thus each chiral center inFormula 1 can exist in either R or S configuration. The definition of R(rectus) and S (sininster) are described in the IUPAC 1974Recommendations, Section E, Fundamental Setereochemistry: The rules canbe found in Pure Appl. Chem 45, 13-30 (1976) and In “Nomenclature ofOrganic Chemistry” pergamon, New York, 1979.

LNA compounds can include an activation group for —OH, —SH, and—NH(R^(H)), respectively. Such activation groups are, for example,selected from optionally substituted O-phosphoramidite, optionallysubstituted O-phosphortriester, optionally substitutedO-phosphordiester, optionally substituted H-phosphonate, and optionallysubstituted O-phosphonate.

B constitutes a natural or non-natural nucleobase and selected amongadenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine,guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil,5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine,2-aminopurine, inosine, diaminopurine, 7-propyne-7-deazaadenine,7-propyne-7-deazaguanine, and 2-chloro-6-aminopurine.

Preferably, the Locked Nucleic Acid (LNA) used in a segmented miRNAmimetic of the invention comprises at least one nucleotide comprises aLocked Nucleic Acid (LNA) unit according any of the Formulas II:

wherein Y is —O—, —S—, —NH—, or N(R^(H)); Z and Z* are independentlyselected among an internucleotide linkage, a terminal group or aprotecting group; and B constitutes a natural or non-natural nucleobase.These exemplary LNA monomers and others, as well as their preparationare described in WO 99/14226 and subsequent applications, WO 00/56746,WO 00/56748, WO 00/66604, WO 00/125248, WO 02/28875, WO 2002/094250 andWO 2003/006475, the disclosure of all of which are incorporated hereinby reference.

As used herein, the term “mimetic” refers to its meaning as is generallyaccepted in the art. The term generally refers to a molecule that isstructurally different from the reference molecule (e.g., thecorresponding naturally-existing molecule or the correspondingnon-segmented mimetic molecule) but is capable of performing one or moreor all of the biological, physiological, and/or chemical functions thatare within the capabilities of the references molecule. The mimetic andthe reference molecule do not have to be functional equivalents but themimetic should be able to perform one or more functions, and exhibitingat least 5% or more, 10% or more, 20% or more, 30% or more, 40% or more,50% or more, 60% or more, 70% or more, 80% or more, or 90% or more ofthe activity of the reference molecule, as measured and compared usingassays or parameters that are suitable to represent the sharedfunction(s). As used herein, a segmented miRNA molecule is a miRNAmimetic when the former shares at least one function with itscorresponding endogenous miRNA. A miRNA mimetic can be a synthetic RNAduplex, such as a segmented miRNA duplex of the invention, avector-encoded hairpin molecule, or other suitable structures designedbased on a corresponding naturally-occurring endogenous miRNA.

The term, “miRNA” or “microRNA” refers to its meaning as is generallyaccepted in the art. The term generally refers to an endogenous shortRNA molecule, which can be isolated or synthetic, which is found ineukaryotes and is involved in RNA-based gene regulation. Arepresentative set of known endogenous miRNA species is described in thepublicly available miRBase sequence database as described inGriffith-Jones er al., Nucleic Acids Research, 2004, 32:D109-D111 andGriffith-Jones et al., Nucleic Acids Research, 2006, 34:D 140-D144,accessible on the World Wide Web at the Wellcome Trust Sanger Institutewebsite. A more selected set of miRNA species are included in Table Iherein. Each mature miRNA is partially complementary to one or moremessenger RNA (mRNA) molecules, which are also called “miRNA targets,”thereby regulating the expression of genes associated with the miRNAtargets.

The term “nick” as used herein refers to a break in an internucleotidelinkage in one or both strands of a segmented miRNA mimetic of theinvention.

The term “non-nucleotide” refers to its meaning as is generally acceptedin the art. The term generally refers to any group or compound which canbe incorporated into a nucleic acid chain in the place of one or morenucleotide units, such as for example but not limitation abasicmoieties, alkyl moieties, polymers such as PEG, peptides, sterols,peptide nucleic acids, and the like.

The term “nucleotide” refers to its meaning as is generally accepted inthe art. The term generally refers to compounds that comprise anucleobase, a sugar, and an internucleoside linkage, e.g., a phosphategroup such as a phosphodiester. The base can be a natural bases(standard), modified bases, or a base analog, as are well known in theart. Such bases are generally located at the 1′ position of a nucleotidesugar moiety. Additionally, the nucleotides can be unmodified ormodified at the sugar, internucleoside linkage, and/or base moiety,(also referred to interchangeably as nucleotide analogs, modifiednucleotides, non-natural nucleotides, non-standard nucleotides andothers; see, for example, U.S. application Ser. No. 12/064,014).

The term “parenteral,” refers to its meaning as is generally accepted inthe art. The term generally includes subcutaneous, intravenous,intramuscular, intraarterial, intraabdominal, intraperitoneal,intraarticular, intraocular or retrobulbar, intraaural, intrathecal,intracavitary, intracelial, intraspinal, intrapulmonary ortranspulmonary, intrasynovial, and intraurethral injection or infusiontechniques.

By “passenger strand” of a segmented miRNA of the invention is meant oneor more distinct nucleic acid sequences or contiguous stretches ofnucleotides capable of forming base pairs (including traditional basepairs and non-traditional base pairs) to one or more non-overlappingcontiguous stretches of nucleotides in the guide strand. The nucleotideswithin each contiguous stretch can be connected by traditionalphosphodiester bonds and/or non-phosphodiester connectors. In addition,the passenger strand of a segmented miRNA can comprise one or morenucleic acid sequences having at least substantial homology, or at leastessential homology, or even perfect homology to a RNA sequence that is atarget of a corresponding naturally-occurring miRNA, such as oneselected from the miRBase, and for example, one selected from Table Iherein.

The terms “patient.” “subject,” “individual” refer to their ordinarymeanings as are generally accepted in the art. The terms generally referto any animal or cells or tissues thereof whether in vitro or in situ,amendable to the methods described herein. They typically refer to anorganism, which is a donor or recipient of explanted cells or the cellsthemselves. They also refer to an organism to which the segmented miRNAsof this disclosure can be administered. In certain non-limitingembodiments, the patient, subject or individual is a mammal or amammalian cell. In other non-limiting embodiments, the patient, subjector individual is a human or a human cell.

The term “phospholipid” refers to its meaning as is generally acceptedin the art. The term generally refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

The term “perfect complementarity” (or “perfectly complement”) as usedherein refers to complete (100%) complementarity within a contiguousregion of double-stranded nucleic acid, such as, for example, between ahexamer or heptamer seed sequence of a miRNA and its complementarysequence in a target mRNA. “Perfectly complementary” can also mean thatall the contiguous residues of a first nucleic acid sequence formhydrogen bonds with the same number of contiguous residues in a secondnucleic acid sequence. For example, 2 or more perfectly complementarynucleic acid strands can have the same number of nucleotides (i.e., havethe same length and form one double-stranded region with or without anoverhang), or have a different number of nucleotides (e.g., one strandcan be shorter but fully contained within a second strand). “Perfectcomplements” can be formed between modified nucleotides and nucleotideanalogs.

The term “perfect homology” (or “perfectly homologous”) as used hereinrefers to complete (100%) homology or “identity” between a referencesequence and a subject nucleic acid sequence. When there is a perfecthomology, the reference and the subject sequences are the same.

The term “phosphorothioate” refers to its meaning as is generallyaccepted in the art. The term generally refers a sulphur substitutedinternucleotide phosphate linkage, but can also refer to internucleotidelinkages selected from the group consisting of: —O—P(O)₂—O—,—O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—,—O—P(O)₂—S—, —OP(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—,—O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,—NR^(H)—CO—NR^(H)—, and/or the internucleotide linkage can be selectedform the group consisting of: —O—CO—O—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—,—O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—, —CH₂—NR^(H)—CO—, —O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—SO₂—CH₂—,—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—CO—, —CH₂—NCH₃—O—CH₂—, where R^(H) isselected from hydrogen and C₁₋₄-alkyl, Suitably, in some embodiments,sulphur (S) containing internucleotide linkages as provided above can bepreferred. Moreover, a segmented miRNA mimetic of the invention cancomprise other linkages or mixtures of different linkages—for example,both phosphate or phosphorothioate linkages, or just phosphate linkages,or other linkages as described herein.

The terms “polynucleotide” and “oligonucleotide” refer to their meaningsas are generally accepted in the art. The terms generally refers to achain of nucleotides. “Nucleic acids” or “nucleic acid molecules” arepolymers of nucleotides. Thus, “nucleic acids” and “polynucleotides” or“oligonucleotides” are interchangeable herein. One skilled in the arthas the general knowledge that nucleic acids are polynucleotides, whichcan be hydrolyzed into monomeric nucleotides. The monomeric nucleotidescan be further hydrolyzed into nucleosides.

The term “protecting group” refers to its meaning as is generallyaccepted in the art. Protection groups of hydroxy substituents comprisessubstituted trityl, such as 4,4′-dimethoxytrityloxy (DMT),4-monomethoxytrityloxy (MMT), and trityloxy, optionally substituted9-(9-phenyl)xanthenyloxy (pixyl), optionally substitutedmethoxytetrahydro-pyranyloxy (mthp), silyloxy such as trimethylsilyloxy(TMS), triisopropylsilyloxy (TIPS)₇ tert-butyldimethylsilyloxy (TBDMS),triethylsilyloxy, and phenyldimethylsilyloxy, tert-butylethers, acetals(including two hydroxy groups), acyloxy such as acetyl or halogensubstituted acetyls.

The term “purine” refers to its meaning as is generally accepted in theart. The term generally refers to conventional purine nucleotides,including those with standard purine bases adenine and guanine. Inaddition, the term “purine” is contemplated to embrace nucleotides withnatural non-standard purine bases or acids, such as N₂-methylguanine,inosine, 2,6-diaminopurine and the like, as well as chemically modifiedbases or “universal bases,” which can be used to substitute for standardurines herein.

The term “pyrimidine” refers to its meaning as is generally accepted inthe art. The term generally refers to conventional pyrimidinenucleotides, including those with standard pyrimidine bases uracil,thymidine and cytosine. In addition, the term pyrimidine is contemplatedto embrace nucleotides with natural non-standard pyrimidine bases oracids, such as 5-methyluracil, 2-thio-5-methyluracil, 4-thiouracil,pseudouracil, dihydrouracil, orotate, 5-methylcytosine, or the like, aswell as a chemically modified bases or “universal bases,” which can beused to substitute for a standard pyrimidine within the nucleic acidmolecules of this disclosure.

The term “RNA” refers to its meaning as is generally accepted in theart. The term generally refers to a molecule comprising at least oneribofuranoside residue, such as a ribonucleotide. The term“ribonucleotide” means a nucleotide with a hydroxyl group at the 2′position of a β-D-ribofuranose moiety. The term refers to adouble-stranded RNA, a single-stranded RNA, an isolated RNA such as apartially purified RNA, an essentially pure RNA, a synthetic RNA, arecombinantly produced RNA, or an altered RNA that differs from anaturally-occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides therein. Such alterations caninclude addition of non-nucleotide material, for example, at one or morenon-terminal nucleotides of an RNA molecule. As such, nucleotides in thesegmented miRNA mimetics of the invention can comprise non-standardnucleotides, such as non-naturally occurring nucleotides, chemicallysynthesized and/or modified nucleotides, or deoxynucleotides. Thealtered RNAs are referred to as “RNA analogs” or “analogs ofnaturally-occurring RNA” containing standard nucleotides (i.e., adenine,cytidine, guanidine, thymidine and uridine), or generally as “modifiedRNA”.

As used herein, the phrase “RNA interference” (also called “RNAi”herein) refers to its meaning as is generally accepted in the art. Theterm generally refers to the biological process of inhibiting,decreasing, or down-regulating gene expression in a cell, and which ismediated by short interfering nucleic acid molecules (e.g., siRNAs,miRNAs, shRNAs), see for example Zamore and Haley, 2005, Science309:1519-1524; Vaughn and Martienssen, 2005, Science 309:1525-1526;Zamore et al., 2000, Cell 101:25-33; Bass, 2001, Nature 411:428-429;Elbashir et al., 2001, Nature 411:494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science 297:1818-1819;Volpe et al., 2002, Science 297:1833-1837; Jenuwein, 2002, Science297:2215-2218; and Hall et al., 2002, Science 297:2232-2237; Hutvagnerand Zamore, 2002, Science 297:2056-60; McManus et al., 2002, RNA8:842-850; Reinhart et al., 2002, Gene & Dev. 16:1616-1626; and Reinhart& Bartel, 2002, Science 297:1831). Additionally, the term “RNAinterference” (or “RNAi”) is meant to be equivalent to other terms usedto describe sequence-specific RNA interference, such aspost-transcriptional gene silencing, translational inhibition,transcriptional inhibition, or epigenetics. For example, segmentedmicroRNA mimetics of the invention can be used to epigenetically silencegenes at either the post-transcriptional level or thepre-transcriptional level. In a non-limiting example, epigeneticmodulation of gene expression by segmented microRNA mimetics of theinvention can result from modification of chromatin structure ormethylation patterns to alter gene expression (see, for example, Verdelet al., 2004, Science 303:672-676; Pal-Bhadra et al., 2004, Science303:669-672; Allshire, 2002, Science 297:1818-1819; Volpe et al., 2002,Science 297:1833-1837; Jenuwein, 2002, Science 297:2215-2218; and Hallet al., 2002, Science 297:2232-2237). In another non-limiting example,modulation of gene expression by segmented microRNA mimetics of theinvention can result from cleavage of RNA (either coding or non-codingRNA) via RISC, or via translational inhibition, as is known in the artor modulation can result from transcriptional inhibition (see forexample Janowski et al., 2005, Nature Chemical Biology 1:216-222).

The term “RNA profile” or “gene expression profile” refers to a set ofdata regarding the expression pattern for one or more gene or geneticmarker in the sample (e.g., a plurality of nucleic acid probes thatidentify one or more markers). In some embodiments, it can be useful toknow whether a cell expresses a particular miRNA endogenously or whethersuch expression is affected under particular conditions or when it is ina particular disease state. Thus in some embodiments of the invention,methods include assaying a cell or a sample containing a cell for thepresence of one or more marker genes or mRNA or other analyte indicativeof the expression level of a gene of interest. Consequently in someembodiments, methods include a step of generating an RNA profile for asample.

As used herein, the term “seed sequence” refers to at least 6consecutive nucleotides within any of nucleotide positions 1 to 10 ofthe 5′-end of a naturally-occurring mature miRNA, such as one selectedfrom those listed in miRBase (http://www.mirbase.org/) as of the filingdate of the present application, and for example, such as one selectedfrom those listed in Table I, wherein the seed sequence nucleotides ofpositions 1 to 8 are capitalized. See, e.g., Brennecke et al., 2005,PLOS Biol. 3(3):e85. In a naturally-occurring miRNA, the seed sequencetypically determines the target mRNA sequence to which the miRNA canbind and provide gene regulation. As such, multiple naturally-occurringmiRNAs can share a seed sequence, or share substantial homology in theseed sequences, and these miRNAs are members of the same miRNA family.

The term “segmented miRNA mimetic” (or “segmented miRNA,”interchangeably) as used herein refers to a miRNA mimetic moleculecomprising at least one discontinuity in the guide strand that iscapable of modulating the expression of a target gene that is alsoregulated by a corresponding naturally-occurring miRNA, such as oneselected from the miRBase as of the filing date of the presentapplication, and for example, such as one selected from SEQ ID NOs:1-1090 of Table I herein. The discontinuity comprises one or more nicks,gaps, substitutions, or insertions. In one aspect, a segmented miRNAmimetic of the invention will mediate gene silencing via an RNAimechanism such as RISC mediated cleavage, translational inhibition, orepigenetic silencing as is known in the art. A segmented miRNA of theinvention can comprise one or more or all ribonucleotides. SegmentedmiRNAs of the invention can also comprise nucleotide and non-nucleotideanalogs as described herein and as otherwise known in the art.

A segmented miRNA mimetic of the invention is said to be“double-stranded” if the molecule has an overall double-strandedconformation. Each of the “strands” is not necessarily continuous, butrather can comprise one or more distinct contiguous stretches ofnucleotides, separated by non-contiguous segments (i.e. gaps, nicks,substitutions, insertions). The strand (including the one or morecontiguous stretches of nucleotides) that comprises or comprisesessentially of a sequence of a corresponding miRNA target is termed the“passenger strand of the segmented miRNA.” The strand (including the oneor more contiguous stretches of nucleotides) that comprises or comprisesessentially of at least a portion (e.g., a stretch of about 5 to about 8nucleotides within the seed sequence) of a corresponding endogenousmiRNA is termed the “guide strand of the segmented miRNA.” Moreover, aguide strand comprising one or more discontinuities (i.e., gaps, nicks,substitutions, insertions) can form a double-stranded RNA complex evenif it is hybridized to a passenger strand that also comprises one ormore discontinuities (i.e., gaps, nicks, substitutions, insertions).When both strands comprise discontinuities, the discontinuities can, incertain embodiments, be arranged in such relative positions with eachother that the segmented miRNA mimetic of the invention maintains agenerally double-stranded conformation, thereby allowing its recognitionby the cellular RNAi machinery. Linkers can be introduced and variousother stabilizing modifications can be applied to confer addedthermodynamic stability. In a specific embodiment, linkers orstabilizing modifications are introduced to a molecule comprisingstructurally overlapping discontinuities, where otherwise thedouble-stranded molecule would have broken into two double-strandedsections if the contiguous stretches adjacent to the overlapping gapsare not connected.

Each segmented miRNA mimetic of the invention can have a correspondingnon-segmented double-stranded miRNA mimetic, where the non-segmentedmimetic comprises all of the contiguous stretches of nucleotides of thesegmented mimetic, and where the non-segmented mimetic and the segmentedmimetic share the same RNA target(s) with their corresponding endogenousmiRNA. Essentially, the segmented miRNA mimetic is designed based on thecorresponding non-segmented miRNA mimetic by deleting certain internalphosphodiester backbone linkages and/or internal nucleotides (i.e., byplacing nicks or gaps) or substituting such nicks or gaps with one ormore non-nucleotide moieties.

It is contemplated that multiple segmented miRNAs, having respectivemultiple corresponding miRNAs, can be applied to a cell. In particularembodiments, two or more segmented miRNAs are introduced to a cell. Acombination of multiple segmented miRNAs can act as one or more pointsof regulation in cellular pathways within the cell, which has aberrantphenotype(s) (i.e., that the cell is a “targeted cell”), and that suchcombination can have increased efficacy for correcting the aberrantphenotype(s) of the targeted cell. If the targeted cell is mixed withnormal cells, it is contemplated that the segmented miRNAs added to thecollection of cells, while providing improved efficacy to correct theaberrant phenotypes of the targeted cell, have minimal adverse effect onthe normal cells.

The term “siRNA” (also “short interfering RNA” or “small interferingRNA”) is given its ordinary meaning as is recognized in the art.

A double-stranded nucleic acid molecule can have strands that are notperfectly complementary, but merely “substantially complementary.” By“substantially complementary” it is meant that the nucleic acid sequenceof the first strand is at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%complementary to the nucleic acid sequence of the second strand. Incertain embodiments, complementary nucleic acid molecules can havewrongly paired bases—that is, bases that cannot form a traditionalWaston-Crick base pair (i.e., forming a hydrogen bond) or othernon-traditional types of base pair (i.e., “mismatched” bases, formed orheld together by non-traditional forces that are not hydrogen bonds).Non-traditional Waston-Crick base pairs are also understood as“non-canonical base pairs,” which is meant any non-Watson Crick basepair, such as mismatches and/or wobble base pairs, including flippedmismatches, single hydrogen bond mismatches, trans-type mismatches,triple base interactions, and quadruple base interactions. Non-limitingexamples of such non-canonical base pairs include, but are not limitedto, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AAN7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CC reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric,CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-iminosymmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, ACamino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AUN1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GAamino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GCcarbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GGcarbonyl-imino, GG N7-amino, GU amino-2-carbonyl. GU carbonyl-imino, GUimino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3, GAcarbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

As used herein, the term “substantially homologous” (or “substantialhomology”) is meant that the subject sequence shares at least 25% (e.g.,at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99%) homologous nucleotides with thenucleotides of the same nucleotide positions in a reference sequence. By“essentially homologous” (or “essential homology”) it is meant that, afirst part of a subject sequence having a number of consecutivenucleotides is identical to a first part of a reference sequence havingthe same number or consecutive nucleotides, whereas the rest of thesubject sequence, which does not overlap with the first part of thesubject sequence, is substantially homologous to the rest of thereference sequence, which does not overlap with the first part of thereference sequence. For example, as used herein, the term “essentiallyhomologous” with regard to miRNA sequences, can refer to the contiguousstretch from the 5′-terminal of the guide strand of a segmented miRNAmimetic of the invention comprising a sequence that is essentiallyhomologous to a sequence, including the seed sequence, of acorresponding naturally-occurring miRNA. For example, the firstcontiguous stretch from the 5′-terminal of the guide strand can comprisea 6 to 7-nucleotide stretch within that is perfectly complementary to a6 to 7-nucleotide stretch of the seed sequence, where the rest of thenucleotides (including nucleotide analogs) in the contiguous stretch canbe at least 50% homologous to the rest of the corresponding endogenousmature miRNA sequence. The comparison of sequences and determination ofpercent homology and/or identity between two sequences can beaccomplished using a mathematical algorithm of Karlin and Altschul(1990, PNAS 87:2264-2268), modified as in Karlin and Altschul (1993,PNAS 90:5873-5877) or by visual inspection.

As used herein, the term “substitute non-nucleotide moieties” refers tochemical moieties that are capable of substituting one or morenucleotides in a segmented miRNA mimetic of the invention. Thesubstitute non-nucleotide moieties can allow for non-traditionalbase-pairing (i.e., not forming traditional hydrogen bonds) between thestrands and contribute to the binding free energy. In certainembodiments, the substitute non-nucleotide moieties of the instantdisclosure are those that are capable of associating or otherwiseinteracting with one or more components of the cellular RNAi machinery,including, for example, the PAZ domain, the PIWI domain and/or otherArgonaute protein domains associated with the RISC.

The term “substitution” as used herein refers to a discontinuity inwhich one or more nucleotide(s) of the otherwise continuous nucleotidesequence of the guide strand and/or passenger strand is replaced withone or more non-nucleotide moieties. Examples of such non-nucleotidemoieties are provided herein and others are provided as is generallyknown to those of skill in the art.

The segmented miRNAs of the invention are typically synthetic. The term“synthetic” as used herein generally refers to nucleic acid moleculesthat are not produced naturally in a cell. In certain aspects, thechemical structure of a synthetic nucleic acid molecule can deviate froma naturally-occurring nucleic acid molecule. On the other hand, asynthesized segmented miRNA can encompass all or part of anaturally-occurring miRNA sequence or a component thereof. Moreover, itis contemplated that, in a specific embodiment, a synthetic segmentedmiRNA mimetic administered to a cell can subsequently be altered orprocessed by the cellular components such that its post-processingstructure or sequence can be identical to the whole or a part of anaturally-occurring miRNA. The difference between a synthetic miRNAmimetic and its corresponding endogenous miRNA, including miRNAprecursors and complements, can comprise missing internal (i.e., atnon-terminal positions) phosphordiester bonds, or missing internalnucleotides, altered types of nucleotides, altered internucleotideconnectors or linkages, or chemically modified nucleotides. In certainaspects, a synthetic segmented miRNA of the invention is an RNA or anRNA analog.

The phrases “target site,” “target sequence,” and “target region,” asused herein, refer to their meanings as generally accepted in the art.The terms generally refer to a sequence within a target nucleic acidmolecule (e.g., target RNA) that is “targeted,” e.g., for cleavagemediated by an RNAi molecule that contains a sequence within itsguide/antisense region that is partially, substantially, or perfectlycomplementary to that target sequence. A “target site” for a miRNAmimetic molecule of the present invention refers to a nucleic acidsequence that is partially, substantially, or perfectly complementary tothe guide strand of the miRNA mimetic. The target site can be within acoding or a non-coding (i.e., untranslated) region of a target RNA. Thetarget site can be the target site for an endogenous miRNA for which thesegmented miRNA molecule is a mimetic, in which case the “target site”can also be referred to as a “miRNA target site” or a “correspondingmiRNA target site.”

Linkers connecting the terminal ends of a segmented miRNA mimetic of theinvention are referred to as “terminal linkers” herein.

The term “therapeutic” refers to its meaning as is generally accepted inthe art. The term generally refers to a treatment and/or prophylaxis. Atherapeutic effect is obtained by suppression, remission, or eradicationof a disease state. In the instant application, the disease state isparticularly referred to as one associated with aberrant biologicalpathways regulated by miRNAs, such as those listed in the miRBase at thetime of filing of this application, and especially those listed in TableI herein. The term “treatment” as used herein is meant to includetherapeutic treatment as well as prophylactic, or suppressive measuresfor diseases or disorders. Thus, for example, the term “treatment”includes the administration of an agent prior to or following the onsetof a disease or disorder thereby preventing or removing all signs of thedisease or disorder. As another example, administration of the agentafter clinical manifestation of the disease to combat the symptoms ofthe diseases is also comprised by the term “treatment.”

As used herein, the term “therapeutically effective amount” refers toits meaning as is generally accepted in the art. The term can refer toan amount of a segmented miRNA that is sufficient to result in adecrease in severity of disease symptoms, an increase in frequency orduration of disease symptom-free periods, or a prevention of impairmentor disability due to the disease, in the subject (e.g., a mammal orhuman) to which it is administered. One of ordinary skill in the artwould be able to determine such therapeutically effective amounts basedon such factors such as the subject's size, the severity of symptoms,and the particular composition or route of administration selected. Forexample, a therapeutically effective amount of a segmented miRNA of theinvention, individually, in combination, or in conjunction with otherdrugs, can be used or administered at a therapeutically effective amountto a subject or by administering to a particular cells under conditionssuitable for treatment, to, for example, decrease tumor size, orotherwise ameliorate symptoms associated with a particular disorder inthe subject.

As used herein, “terminals” or “terminal ends” refers to the ultimateends at the first 5′-nucleotide or the first 3′-nucleotide of a givenstrand. Substitutions of such terminal ends can be selectedindependently from hydrogen, azido, halogen, cyano, nitro, hydroxy,Prot-O—, Act-O—, mercapto, Prot-S—, Act-S—, C₁₋₆-alkylthio, amino.Prot-N(R^(H))—, Act-N(R^(H))—, mono- or di(C₁₋₆-alkyl)amino, optionallysubstituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl, optionallysubstituted C₂₋₆-alkenyl, optionally substituted C₂₋₆-alkenyloxy,optionally substituted C₂₋₆-alkynyl, optionally substitutedC₂₋₆-alkynyloxy, monophosphate, monothiophosphate, diphosphate,dithiophosphate triphosphate, trithiophosphate, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl,Prot-O—CH₂—, Act-O—CH₂—, aminomethyl, Prot-N(R^(H))—CH₂—,Act-N(R^(H))—CH₂—, carboxy methyl, sulphonomethyl, where Prot is aprotection group for —OH, —SH, and —NH(R^(H)), respectively, Act is anactivation group for —OH, —SH, and —NH(R^(H)), respectively, and R^(H)is selected from hydrogen and C₁₋₆-alkyl.

Linkers connecting the terminal ends of a segmented miRNA mimetic of theinvention are referred to as “terminal linkers” herein.

The term “universal base” refers to its meaning as is generally acceptedin the art. The term generally refers to nucleotide base analogs thatform base pairs with each of the standard DNA/RNA bases with littlediscrimination among them, and is recognized by intracellular enzymes.See, e.g., Loakes et al., J. Mol. Bio. 1997, 270:426-435. Non-limitingexamples of universal bases include C-phenyl, C-naphthyl and otheraromatic derivatives, inosine, azole carbozamides, and nitroazolederivatives such as 3′-nitropyrrole, 4-nitroindole, 5-nitroindole, and6-nitroindole as known in the art. See, e.g., Loakes, 2001 Nucleic AcidsRes. 29:2437.

A “vector” refers to its meaning as is generally accepted in the art.The term generally refers to a replicon, such as a plasmid, phagemid,cosmid, baculovirus, bacmid, bacterial artificial chromosome (BAC),yeast artificial chromosome (YAC), as well as other bacterial, yeast, orviral vectors, to which another nucleic acid segment can be operativelyinserted so as to bring about replication or expression of the insertedsegment. “Expression vector” refers to a vector comprising expressioncontrol sequences operatively linked to a nucleotide sequence to beexpressed. An expression vector comprises sufficient cis-acting elementsfor expression; other elements for expression can be supplied by thehost cell or in an in vitro expression system. Expression vectorsinclude all those known in the art, such as cosmids, plasmids (e.g.,naked or contained in liposomes), and viruses (e.g., lentiviruses,retroviruses, adenoviruses, and adeno-associated viruses).

Any concentration range, percentage range, ratio range, or integer rangeis to be understood to include the value of any integer within therecited range, and when appropriate, fractions thereof (such as onetenth and one hundredth of an integer), unless otherwise indicated.

Other objects, features and advantages of the present invention willbecome apparent from the detailed description. It should be understood,however, that the detailed description and the specific examples, whileindicating specific embodiments of the invention, are given by way ofillustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from the detailed description.

A Segmented miRNA of the Invention

The instant disclosure provides a segmented miRNA mimetic molecule(segmented miRNA mimetic) that is double-stranded comprising a passengerstrand and a distinct guide strand, wherein at least the guide strandincludes one of more discontinuities and wherein the passenger strandand the guide strand each independently comprises, in sum, about 12 toabout 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, or 27) nucleotides, and the mimetic molecule comprises, in sum,about 10 to about 26 (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, or 27) base pairs. In one aspect, asegmented miRNA of the invention comprises 2 or more (e.g., 2, 3, or 4)distinct contiguous stretches of nucleotides in the guide strand. A“contiguous stretch of nucleotides” can comprise as little as 2nucleotides, according to the present invention. In another aspect, asegmented miRNA of the invention comprises 2 or more (e.g., from 2, 3,4, 5, 6, 7, 8, 9, or 10) or more distinct contiguous stretches ofnucleotides in each of the passenger strand and the guide strand. Thedistinct contiguous stretches of nucleotides are arranged such thattheir complementary sequences located on the opposite strand or within acorresponding miRNA target sequence do not overlap. Within each of thecontiguous stretches of nucleotides, the nucleotides are connected byphosphodiester bonds and/or non-phosphodiester connectors. The distinctcontiguous stretches of nucleotides in a given strand are arranged from5′- to 3′-, and the each pair of neighboring stretches can be separatedby a nick, gap, substitution, or insertion.

In one embodiment, a first discontinuity in the passenger strand and asecond discontinuity in the guide strand of a segmented miRNA mimetic donot overlap provided that RNAi activity against one or more miRNAtargets is maintained.

In another embodiment, a first discontinuity in the passenger strand anda second discontinuity in the guide strand of a segmented miRNA mimeticpartially overlap provided that RNAi activity against one or more miRNAtargets is maintained.

In another embodiment, a first discontinuity in the passenger strand anda second discontinuity in the guide strand of a segmented miRNA mimeticoverlap completely provided that RNAi activity against one or more miRNAtargets is maintained. For example, overlapping nicks or gaps can resultin a miRNA mimetic molecule that is no longer able to associate intoduplex form. One of skill in the art will readily appreciate that suchdesigns are to be avoided in order to maintain miRNA medicated RNAiactivity.

In one embodiment, a first nick in the passenger strand and a secondnick in the guide strand of a segmented miRNA mimetic do not overlap.

In one embodiment, a first gap in the passenger strand and a second gapin the guide strand of a segmented miRNA mimetic do not overlap by atleast one complementary nucleotide position.

In one aspect, a segmented miRNA mimetic molecule of the invention canbe represented or depicted by Formula III:

wherein the molecule comprises a passenger strand and a guide strand,where each line shown in the Formula and its adjacent “N” represent acontiguous stretch of nucleotides, each of “X1,” “X2” and “X3” representthe number of nucleotide positions in each stretch, “G/N” represents adiscontinuity in the guide strand, “Y1” represents a number ofnucleotide positions in the discontinuity, and each group of dashedlines “

” and its adjacent “(W)” represents a terminal overhang that isoptionally present or absent, and each of “Z1” and “Z2” represents thenumber of overhanging nucleotides.

With reference to Formula III, in one embodiment, X1 is an integer fromabout 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, or 27), X2 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X3 isan integer from about 6 to about 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y1 is an integerfrom 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11),provided that the sum of X2, X3 and Y1 is an integer from about 16 toabout 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27).In certain embodiments the discontinuity in the guide strand is a nick.In certain embodiments the discontinuity in the guide strand is a gap.In certain embodiments the discontinuity in the guide strand is asubstitution. In certain embodiments the discontinuity in the guidestrand is an insertion. In one embodiment, there is no 3′-terminaloverhanging nucleotides present (i.e., blunt-ended) in the passengerstrand, in the guide strand, or in either strand, i.e., wherein Z1, Z2,or both Z1 and Z2 are 0. In another embodiment, there are one or more3′-terminal overhanging nucleotides present in the passenger strand,wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In a furtherembodiment, there are one or more 3′-terminal overhanging nucleotidespresent in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1,2, 3, 4, or 5). In yet another embodiment, there are one or more3′-terminal overhanging nucleotides present in both the passenger strandand the guide strand, wherein Z1 and Z2 are independently about 1 toabout 5 (e.g., 1, 2, 3, 4, or 5).

With reference to Formula III, in one embodiment, X1 is an integer fromabout 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, or 27), X2 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X3 isan integer from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y1is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or 11), provided that the sum of X2, X3 and Y1 is an integer fromabout 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, or 27). In certain embodiments the discontinuity in the guide strandis a nick. In certain embodiments the discontinuity in the guide strandis a gap. In certain embodiments the discontinuity in the guide strandis a substitution. In certain embodiments the discontinuity in the guidestrand is an insertion. In one embodiment, there is no 3′-terminaloverhanging nucleotides present (i.e., blunt-ended) in the passengerstrand, in the guide strand, or in either strand, i.e., wherein Z1, Z2,or both Z1 and Z2 are 0. In another embodiment, there are one or more3′-terminal overhanging nucleotides present in the passenger strand,wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In a furtherembodiment, there are one or more 3′-terminal overhanging nucleotidespresent in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1,2, 3, 4, or 5). In yet another embodiment, there are one or more3′-terminal overhanging nucleotides present in both the passenger strandand the guide strand, wherein Z1 and Z2 are independently about 1 toabout 5 (e.g., 1, 2, 3, 4, or 5).

In another aspect, a segmented miRNA mimetic molecule of the inventioncan be represented or depicted by Formula IV:

wherein the molecule comprises a passenger strand and a guide strand,where each line in the Formula and its adjacent “N” represent acontiguous stretch of nucleotides, each of “X1,” “X2,” “X3” and “X4”represents a number of nucleotide positions in each stretch, “P/N”represents a discontinuity in the passenger strand, “G/N” represents adiscontinuity in the guide strand, “P/N” represents a discontinuity inthe passenger strand, each of “Y1” and “Y2” represents a number ofnucleotide positions in the discontinuity, and each group of dashedlines “

” and its adjacent “(W)” represents a terminal overhang that isoptionally present or absent, and each of “Z1” and “Z2” represents thenumber of overhanging nucleotides.

With reference to Formula IV, in one embodiment, X1 and X2 are integersindependently from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25), Y1 isan integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11), provided that the sum of X1, X2 and Y1 is an integer from about16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or27), X3 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X4 is aninteger from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y2 is aninteger from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or11), provided that the sum of X3, X4 and Y2 is about 16 to about 26(e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). Incertain embodiments the discontinuity in the guide and/or passengerstrand is a nick. In certain embodiments the discontinuity in the guideand/or passenger strand is a gap. In certain embodiments thediscontinuity in the guide and/or passenger strand is a substitution. Incertain embodiments the discontinuity in the guide and/or passengerstrand is an insertion. In certain embodiments, there is no 3′-terminaloverhanging nucleotides present (i.e., blunt-ended) in the passengerstrand, in the guide strand, or in either strand, i.e. wherein Z1, Z2,or both Z1 and Z2 are 0. In another embodiment, there are one or more3′-terminal overhanging nucleotides present in the passenger strand,wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certainembodiments, there are one or more 3′-terminal overhanging nucleotidespresent in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1,2, 3, 4, or 5). In yet another embodiment, there are one or more3′-terminal overhanging nucleotides present in both the passenger strandand the guide strands, wherein Z1 and Z2 are independently about 1 toabout 5 (e.g., 1, 2, 3, 4, or 5).

With reference to Formula IV, in one embodiment, X1 and X2 are integersindependently from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25), Y1 isan integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11), provided that the sum of X1, X2 and Y1 is an integer from about16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or27), X3 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X4 is aninteger from about 6 to about 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y2 is an integerfrom 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11),provided that the sum of X3, X4 and Y2 is about 16 to about 26 (e.g.,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certainembodiments the discontinuity in the guide and/or passenger strand is anick. In certain embodiments the discontinuity in the guide and/orpassenger strand is a gap. In certain embodiments the discontinuity inthe guide and/or passenger strand is a substitution. In certainembodiments the discontinuity in the guide and/or passenger strand is aninsertion. In certain embodiments, there is no 3′-terminal overhangingnucleotides present (i.e., blunt-ended) in the passenger strand, in theguide strand, or in either strand, i.e. wherein Z1, Z2, or both Z1 andZ2 are 0. In another embodiment, there are one or more 3′-terminaloverhanging nucleotides present in the passenger strand, wherein Z1 isabout 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments,there are one or more 3′-terminal overhanging nucleotides present in theguide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).In yet another embodiment, there are one or more 3′-terminal overhangingnucleotides present in both the passenger strand and the guide strands,wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3,4, or 5).

In yet another aspect, a segmented miRNA mimetic molecule of theinvention can be represented or depicted by Formula V:

wherein the molecule comprises a passenger strand and a guide strand,where each line in the Formula and its adjacent “N” represent acontiguous stretch of nucleotides, each of “X1,” “X2,” “X3” and “X4”represents the number of nucleotide positions in each stretch, each“G/N” represents a discontinuity in the guide strand, each of “Y1” and“Y2” represents the number of nucleotide positions in the discontinuity,and each group of dashed lines “

” and its adjacent “(W)” represents a terminal overhang that isoptionally present or absent, and each of “Z1” and “Z2” represents thenumber of overhanging nucleotides; wherein X1 is an integer from about12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, or 27), X2 and X3 are each independently an integer fromabout 1 to about 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, or 17), X4 is an integer from about 6 to about 22 (e.g., 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23),Y1 and Y2 are each independently an integer from 0 to about 10 (e.g., 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X2, X3,X4, Y1 and Y2 is an integer from about 10 to about 26 (e.g., 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). Incertain embodiments the discontinuity in the guide and/or passengerstrand is a nick. In certain embodiments the discontinuity in the guideand/or passenger strand is a gap. In certain embodiments thediscontinuity in the guide and/or passenger strand is a substitution. Incertain embodiments the discontinuity in the guide and/or passengerstrand is an insertion. In certain embodiments, there is no 3′-terminaloverhanging nucleotides present (i.e., blunt-ended) in the passengerstrand, in the guide strand, or in either strand, wherein Z1, Z2, orboth Z1 and Z2 are 0. In another embodiment, there are one or more3′-terminal overhanging nucleotides present in the passenger strand,wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certainembodiments, there are one or more 3′-terminal overhanging nucleotidespresent in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1,2, 3, 4, or 5). In yet another embodiment, there are one or more3′-terminal overhanging nucleotides present in both the passenger strandand the guide strands, wherein Z1 and Z2 are independently about 1 toabout 5 (e.g., 1, 2, 3, 4, or 5).

In a further aspect, a segmented miRNA mimetic molecule of the inventioncan be represented or depicted by Formula VI:

wherein the molecule comprises a passenger strand and a guide strand,where each line in the Formula and its adjacent “N” represent acontiguous stretch of nucleotides, each of “X1,” “X2,” “X3,” “X4” and“X5” represents the number of nucleotide positions in each stretch,“P/N” represents a discontinuity in the passenger strand, “G/N”represents a discontinuity in the guide strand, each “P/N” represents adiscontinuity in the passenger strand, each of “Y1,” “Y2” and “Y3”represents the number of nucleotide positions in the discontinuity, andeach group of dashed lines “

” and its adjacent “(W)” represents a terminal overhang that isoptionally present or absent, and each of “Z1” and “Z2” represents thenumber of overhanging nucleotides; wherein X1, X2, and X3 are eachindependently an integer from about 2 to about 22 (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23),Y1 and Y2 are each independently an integer from 0 to about 10 (e.g., 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2,X3, Y1 and Y2 is an integer from about 12 to about 26 (e.g., 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X4 is aninteger from about 1 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X5 is an integer fromabout 6 to about 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25), Y3 is an integer from 0 to about10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that thesum of X4, X5 and Y3 is an integer from about 10 to about 26 (e.g., 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27).In certain embodiments the discontinuity in the guide and/or passengerstrand is a nick. In certain embodiments the discontinuity in the guideand/or passenger strand is a gap. In certain embodiments thediscontinuity in the guide and/or passenger strand is a substitution. Incertain embodiments the discontinuity in the guide and/or passengerstrand is an insertion. In certain embodiments, there is no 3′-terminaloverhanging nucleotides present (i.e., blunt-ended) in the passengerstrand, in the guide strand, or in either strand, wherein Z1, Z2, orboth Z1 and Z2 are 0. In another embodiment, there are one or more3′-terminal overhanging nucleotides present in the passenger strand,wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certainembodiments, there are one or more 3′-terminal overhanging nucleotidespresent in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1,2, 3, 4, or 5). In yet another embodiment, there are one or more3′-terminal overhanging nucleotides present in both the passenger strandand the guide strands, wherein Z1 and Z2 are independently about 1 toabout 5 (e.g., 1, 2, 3, 4, or 5).

In a further aspect, a segmented miRNA mimetic molecule of the inventioncan be represented or depicted by Formula VII:

wherein the molecule comprises a passenger strand and a guide strand,where each line in the Formula and its adjacent “N” represent acontiguous stretch of nucleotides, each of “X1,” “X2,” “X3,” “X4,” “X5”and “X6” represents the number of nucleotide positions in each stretch,each “P/N” represents a discontinuity in the passenger strand, each“G/N” represents a discontinuity in the guide strand, each of “Y1,”“Y2,” “Y3” and “Y4” represents the number of nucleotide positions in thediscontinuity, and each group of dashed lines “

” and its adjacent “(W)” represents a terminal overhang that isoptionally present or absent, and each of “Z1” and “Z2” represents thenumber of overhanging nucleotides; wherein X1, X2, and X3 are eachindependently an integer from about 2 to about 22 (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23),Y1 and Y2 are each independently an integer from 0 to about 10 (e.g., 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2,X3, Y1 and Y2 is an integer from about 12 to about 26 (e.g., 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X4 and X5are each independently an integer from about 1 to about 16 (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17), X6 is aninteger from about 7 to about 22 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, or 23), Y3 and Y4 are independently aninteger from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or11), provided that the sum of X4, X5, X6, Y3 and Y4 is about 10 to about26 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, or 27). In certain embodiments the discontinuity in the guideand/or passenger strand is a nick. In certain embodiments thediscontinuity in the guide and/or passenger strand is a gap. In certainembodiments the discontinuity in the guide and/or passenger strand is asubstitution. In certain embodiments the discontinuity in the guideand/or passenger strand is an insertion. In certain embodiments, thereis no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) inthe passenger strand, in the guide strand, or in either strand, whereinZ1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one ormore 3′-terminal overhanging nucleotides present in the passengerstrand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). Incertain embodiments, there are one or more 3′-terminal overhangingnucleotides present in the guide strand, wherein Z2 is about 1 to about5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one ormore 3′-terminal overhanging nucleotides present in both the passengerstrand and the guide strands, wherein Z1 and Z2 are independently about1 to about 5 (e.g., 1, 2, 3, 4, or 5).

In a further aspect, a segmented miRNA mimetic molecule of the inventioncan be represented or depicted by Formula VIII:

wherein the molecule comprises a passenger strand and a guide strand,where each line in the Formula and its adjacent “N” represent acontiguous stretch of nucleotides, each of “X1,” “X2,” “X3” and “X4”represents the number of nucleotide positions in each stretch, “P/N”represents a discontinuity in the passenger strand, each “G/N”represents a discontinuity in the guide strand, “P/N” represents adiscontinuity in the passenger strand, each of “Y1” and “Y2” representsthe number of nucleotide positions in the discontinuity, and each groupof dashed lines “

” and its adjacent “(W)” represents a terminal overhang that isoptionally present or absent, and each of “Z1” and “Z2” represents thenumber of overhanging nucleotides; wherein X1 and X2 are eachindependently an integer from about 2 to about 24 (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24or 25). Y1 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11), provided that the sum of X1, X2 and Y1 is aninteger from about 12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, or 27), X3 and X4 are each independentlyan integer from about 1 to about 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, or 17), X5 is an integer from about 6 toabout 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25), Y2 and Y3 are each independently an integerfrom 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11),provided that the sum of X3, X4, X5, Y2 and Y3 is an integer from about10 to about 26 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity inthe guide and/or passenger strand is a nick. In certain embodiments thediscontinuity in the guide and/or passenger strand is a gap. In certainembodiments the discontinuity in the guide and/or passenger strand is asubstitution. In certain embodiments the discontinuity in the guideand/or passenger strand is an insertion. In certain embodiments, thereis no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) inthe passenger strand, in the guide strand, or in either strand, whereinZ1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one ormore 3′-terminal overhanging nucleotides present in the passengerstrand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). Incertain embodiments, there are one or more 3′-terminal overhangingnucleotides present in the guide strand, wherein Z2 is about 1 to about5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one ormore 3′-terminal overhanging nucleotides present in both the passengerstrand and the guide strands, wherein Z1 and Z2 are independently about1 to about 5 (e.g., 1, 2, 3, 4, or 5).

At least one of the 2 or more contiguous stretches of nucleotides in theguide strand of a segmented miRNA mimetic molecule of Formula III, IV,V, VI, VII, or VIII comprises a sequence that is substantially,essentially or perfectly homologous (e.g., at least 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% homologous) to anucleotide sequence of a naturally-occurring endogenous miRNA, such asone selected from miRBase as of the filing date of the presentinvention, and for example, one selected from SEQ ID NOs: 1-1090 ofTable I herein. In certain embodiments, the first contiguous stretch ofnucleotides from the 5′-end of the guide strand comprises at least 6(e.g., 6, 7, or 8) consecutive nucleotides that are identical (orperfectly homologous) to a 6, 7, or 8-nucleotide sequence within theseed sequence of a naturally-occurring miRNA, such as one selected fromTable I (wherein the seed sequence nucleotides are capitalized). Inanother embodiment, at least 2 of the 2 or more contiguous stretches ofnucleotides in the guide strand of a segmented miRNA mimetic of FormulaIII-VIII comprise sequences that are substantially, essentially, orperfectly homologous (at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100% homologous) to non-overlapping regions of anaturally-occurring endogenous miRNA. In yet another embodiment, all ofthe contiguous stretches of nucleotides in the guide strand comprisesequences that are substantially, essentially, or perfectly homologous(e.g., at least at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100% homologous) to non-overlapping regions of anaturally-occurring endogenous miRNA. Each of the 1 or more contiguousstretches of nucleotides in the passenger strand of a segmented miRNAmimetic molecule of Formula III, IV, V, VI, VII, or VIII comprises asequence that is substantially or perfectly complementary (e.g., atleast 25, 20, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100% complementary) to a non-overlapping region of a naturally-occurringendogenous miRNA, such as one selected from miRBase as of the filingdate of the present invention, and for example, one selected from SEQ IDNOs: 1-1090 of Table I herein.

At least one of the 2 or more contiguous stretches of nucleotides in theguide strand of a segmented miRNA mimetic molecule of Formula III, IV,V, VI, VII, or VIII comprises sequence having 6 or more (e.g., 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or26) contiguous nucleotides of a naturally-occurring endogenous miRNA,such as one selected from miRBase as of the filing date of the presentinvention, and for example, one selected from SEQ ID NOs: 1-1090 ofTable I herein. In certain embodiments, the first contiguous stretch ofnucleotides from the 5′-end of the guide strand comprises at least 6(e.g., 6, 7, or 8) consecutive nucleotides of a seed sequence of anaturally-occurring miRNA, such as one selected from Table I (whereinthe seed sequence nucleotides are capitalized). In another embodiment,at least 2 of the 2 or more contiguous stretches of nucleotides in theguide strand of a segmented miRNA mimetic of Formula III-VIII comprisesequence having 6 or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) contiguous nucleotidesnon-overlapping regions of a naturally-occurring endogenous miRNA. Inyet another embodiment, all of the contiguous stretches of nucleotidesin the guide strand comprises sequence having 6 or more (e.g., 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or26) contiguous nucleotides to non-overlapping regions of anaturally-occurring endogenous miRNA. Each of the 1 or more contiguousstretches of nucleotides in the passenger strand of a segmented miRNAmimetic molecule of Formula III, IV, V, VI, VII, or VIII comprises asequence capable of forming 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26) basepairs to a non-overlapping region of a naturally-occurring endogenousmiRNA, such as one selected from miRBase as of the filing date of thepresent invention, and for example, one selected from SEQ ID NOs: 1-1090of Table I herein.

In one aspect, a segmented miRNA mimetic of the invention comprises twoseparate strands, a guide strand and a separate passenger strand,wherein the strands are not connected to each other at either the 5′ orthe 3′ terminal ends by a linker. Linkers connecting the terminal endsof a segmented miRNA mimetic of the invention are referred to as“terminal linkers” herein. In another aspect, one or both terminal endsof a segmented miRNA mimetic molecule can be connected or linkedtogether by a terminal nucleotide and/or non-nucleotide linker. Incertain embodiments, either or both ends of the passenger strand and theguide strand of a segmented miRNA mimetic of the invention can becovalently linked by a terminal nucleotide and/or non-nucleotide linkeras described herein and known in the art.

One or more substitutions or insertions can be present in the absence ofany terminal linkers, as described above. Alternatively, one or moresubstitutions or insertions can be present in a given molecule with oneor more terminal linkers.

One or more or all of nucleotides of each of the contiguous stretches ofnucleotides can be ribonucleotides, modified ribonucleotides, orsuitable nucleotide analogs. Incorporation of nucleotide analogs, suchas various known sugar, base, and backbone modifications, and LNAmonomer units into disrupted strands can significantly enhance serumstability and prolong target knockdown or expression regulatory effects.The segmented miRNA mimetic molecules of the present invention canfunctionally accommodate and are compatible with various chemicalmodifications, in various combinations and juxtapositions, and tovarying degrees. For example, from one to all (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2526, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, or 52) of the ribonucleotides of thesegmented miRNA mimetics of the invention can be modified. The improvedproperties conferred by the functionally compatible chemicalmodifications to the sugar, base and/or backbone, or by includingsuitable nucleotide analog residues, are of particular importance forapplication of these segmented miRNA mimetic molecules in vivo, forexample, for use as a therapeutic agent or as a functional genomic tool.

In certain embodiments, a segmented miRNA mimetic molecule of thepresent invention can comprise a 3′-terminal overhang in its passengerstrand, guide strand, or both the passenger and guide strands. The“overhang” nucleotides are unpaired and single stranded regions locatedat the terminal ends of an otherwise generally double-stranded nucleicacid molecule. An exemplary segmented miRNA mimetic of the invention cancomprise a 3′-terminal overhang of 1 to 5 nucleotides (e.g., 1, 2, 3, 4,or 5 nucleotides) in the passenger strand, the guide strand, or both thepassenger and the guide strands. In alternative embodiments, a segmentedmiRNA mimetic of the present invention can be blunt-ended (i.e.,comprising no terminal overhang nucleotides) at either or both terminalends.

In a further aspect, the segmented miRNA mimetics of the invention,according to any of the embodiments herein, are capable of participatingin RNAi against endogenous RNA targets of their correspondingnaturally-occurring miRNAs. The inhibition of the miRNA target can beachieved via the standard miRNA-specific interference mechanism. Forexample, the inhibition of the miRNA target can be by interaction (e.g.,base-paring, binding, etc.) with the untranslated mRNA region, withwhich the corresponding endogenous miRNA interacts, which effectuatesthe translational regulation of one or more downstream genes. Or, theinhibition of the miRNA target can be achieved via an siRNA-likeinterference mechanism wherein the binding of the miRNA target by theguide strand of the segmented miRNA mimetic results in the cleavage ofthe untranslated miRNA target.

Modified Segmented miRNA Mimetics

The introduction of modified nucleotide analogs into segmented miRNAmimetic molecules of the invention provides a tool for overcomingpotential limitations of in vivo stability and bioavailability inherentto native RNA molecules (i.e., having standard nucleotides) that areexogenously delivered. In certain embodiments, the use of modifiedsegmented miRNA mimetics of this disclosure can enable achievement of agiven therapeutic effect at a lower dose since these molecules can bedesigned to have an increased melting temperature or half-life in asubject or biological samples (e.g., serum). Furthermore, certainmodifications can be used to improve the bioavailability of segmentedmiRNA mimetics by targeting particular cells or tissues or improvingcellular uptake of the segmented miRNA mimetics. Therefore, even if theactivity of a segmented miRNA mimetic of this disclosure is somewhatreduced (e.g., by less than about 20%, or 30%, or even 40%) as comparedto an unmodified segmented miRNA mimetic of the same structure, theoverall activity of the modified segmented miRNA mimetic can be greaterthan that of its native counterpart due to improved stability ordelivery of the molecule. Modified segmented miRNA mimetics can alsominimize the possibility of activating an interferon response in, forexample, humans.

In certain embodiments, segmented miRNA mimetics of the inventioncomprise ribonucleotides at about 1 or more (e.g. 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or26) of the nucleotide positions in one strand, in each strand, or anycombination thereof.

In related embodiments, a segmented miRNA mimetic according to theinstant disclosure comprises one or more natural or syntheticnon-standard nucleotides. In related embodiments, the non-standardnucleotide is one or more deoxyuridine, L- or D-locked nucleic acid(LNA) molecule (e.g., a 5-methyluridine LNA) or substituted LNA (e.g.,having a pyrene), or a universal-binding nucleotide, or a G clamp, orany combination thereof. In certain embodiments, the universal-bindingnucleotide can be C-phenyl, C-naphthyl, inosine, azole carboxamide,1-β-D-ribofuranosyl-4-nitro indole, 1-β-D-ribofuranosyl-5-nitroindole,1-β-D-ribofuranosyl-6-nitroindole, or1-β-D-ribofuranosyl-3-nitropyrrole.

Modified nucleotides, which can be present in either or both thepassenger and the guide strands of a segmented miRNA mimetic of theinvention, comprise modified nucleotide analogs having characteristicssimilar to natural or standard ribonucleotides. For example, thisdisclosure features segmented miRNA mimetics comprising nucleotideshaving a Northern conformation (see, e.g., Northern pseudorotationcycle, Saenger, Springer-Verlag ed., 1984), which are known topotentially impart resistant to nuclease degradation while maintainingthe capacity to mediate RNAi, at least when applied to siRNA molecules.Exemplary nucleotides having a Northern configuration include lockednucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-methoxyethyl (MOE)nucleotides, 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides,2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, 5-methyluridines,or 2′-O-methyl nucleotides). In any of these embodiments, one or moresubstituted or modified nucleotides can be a G clamp (e.g., a cytosineanalog that forms an additional hydrogen bond to guanine, such as9-(aminoethoxy)phenoxazine). See, e.g., Lin and Mateucci, 1998 J. Am.Chem. Soc. 720:8531).

In certain embodiments, a segmented miRNA mimetic of the inventioncomprises an overhang of 1 to 5 nucleotides. The overhang can compriseone or more 2′-O-alkyl modifications or locked nucleic acid (LNAs) asdescribed herein or otherwise known in the art. In certain embodiments,a segmented miRNA mimetic of the invention can comprise one or more3′-end 2′-O-alkyl or LNA at one or more of the internal terminals. A2′-O-alkyl or LNA can also be present at positions that are not in thegaps, near the nicks or at the terminal ends of a segmented miRNAmimetic. In any of the embodiments of segmented miRNA mimetics, the3′-terminal overhangs, if present, can comprise chemically-modifiednucleotides that are modified at a nucleic acid sugar, base, orbackbone. In any of the embodiments of segmented miRNA mimetics, the3′-terminal nucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of segmented miRNA mimetics,the 3′-terminal nucleotide overhangs can comprise one or more acyclicnucleotides.

In certain embodiments, the 5′-terminal end of the passenger strand orguide strand of a segmented miRNA mimetic of the invention isphosphorylated. In any of the embodiments of segmented miRNA mimeticsdescribed herein, the segmented miRNA can further comprise a terminalphosphate group, such as a 5′-phosphate (see Martinez et al., 2002 Cell110:563; Schwarz et al., 2002 Mole. Cell 70:537) or a 5′3′-diphosphate.

In certain embodiments, a segmented miRNA mimetic comprises one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, or 26) 2′-sugar substitutions in one strandor independently each strand, such as a 2′-deoxy, 2′-O-2-methoxyethyl,2′-O-methoxyethyl, 2′-O-methyl, 2′-halogen (e.g., 2′-fluoro),2′-O-allyl, or the like, or any combination thereof. In still furtherembodiments, a segmented miRNA mimetic comprises a terminal capsubstituent at one or more terminal ends, internal ends, or both, of thepassenger strand and/or the guide strands, such as, for example, analkyl, abasic, deoxy abasic, glyceryl, dinucleotide, acyclic nucleotide,inverted deoxynucleotide moiety, or any combination thereof. In certainembodiments, at least one or more 5′-terminal-end or 5′-internal-endribonucleotides of the passenger strand have 2′-sugar substitutions. Incertain other embodiments, at least one or more 5′-terminal-end or5′-internal-end ribonucleotides of the guide strand have 2′-sugarsubstitutions. In certain embodiments, at least one or more5′-terminal-end or 5′-internal-end ribonucleotides of the passengerstrand and the guide strand have 2′-sugar substitutions.

In other embodiments, a segmented miRNA mimetic comprises one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, or 26) substitutions in the sugar in onestrand or independently each strand, including any combination ofribosyl, 2′-deoxyribosyl, a tetrofuranosyl (e.g., L-α-threofuranosyl), ahexopyranosyl (e.g., β-allopyranosyl, β-altropyranosyl andβ-glucopyranosyl), a pentopyranosyl (e.g., β-ribopyranosyl,α-lyxopyranosyl, β-xylopyranosyl and α-arabinopyranosyl), a carbocyclicanalog, a pyranose, a furanose, a morpholino, or analogs or derivativesthereof.

In yet other embodiments, a segmented miRNA mimetic comprises at leastone (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, or 26) modified internucleoside linkagesin one strand or independently each strand, such as independently aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl phosphonate, alkylphosphonate, 3′-alkylene phosphonate, 5′-alkylene phosphonate, chiralphosphonate, phosphonoacetate, thiophosphonoacetate, phosphinate,phosphoramidate, 3′-amino phosphoramidate, aminoalkylphosphoramidate,thionophosphoramidate, thionoalkylphosphonate,thionoalkylphosphotriester, selenophosphate, boranophosphate linkage, orany combination thereof.

A modified internucleotide linkage, as described herein, can be presentin one or more strands of a segmented miRNA mimetic, for example, in thepassenger strand, the guide strand, or in both strands. A segmentedmiRNA mimetic can comprise one or more modified internucleotide linkagesat the 3′-terminal end, the 5′-terminal end, or both of the 3′-terminaland 5′-terminal ends of the passenger strand, the guide strand, or bothstrands. In certain embodiments, a segmented miRNA mimetic of theinvention has one modified internucleotide linkage at the 3′-terminalend, such as a phosphorothioate linkage. An exemplary segmented miRNAmimetic comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages in either strand. Anotherexemplary segmented miRNA mimetic comprises about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more phosphorothioate internucleotide linkages in bothstrands. A further exemplary segmented miRNA mimetic comprises about 1to about 5 or more consecutive phosphorothioate internucleotide linkagesat for example, the 5′-terminal end of its passenger strand, the5′-terminal end of its guide strand, both the 5′-terminal ends of bothstrands, or for example, at one or more of the 5′-internal ends. In yetanother exemplary segmented miRNA mimetic, there can be one or morepyrimidine phosphorothioate internucleotide linkages in the passengerstrand and/or the guide strand. In a further exemplary segmented miRNAmimetic, there can be one or more purine phosphorothioateinternucleotide linkages in the passenger strand and/or the guidestrand.

Many exemplary modified nucleotide bases or analogs thereof useful insegmented miRNA mimetics of the instant disclosure include5-methylcytosine; 5-hydroxymethylcytosine; xanthine; hypoxanthine;2-aminoadenine; 6-methyl, 2-propyl, or other alkyl derivatives ofadenine and guanine; 8-substituted adenines and guanines (e.g., 8-aza,8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, or the like);7-methyl, 7-deaza, and 3-deaza adenines and guanines; 2-thiouracil;2-thiothymine; 2-thiocytosine; 5-methyl, 5-propynyl, 5-halo (e.g.,5-bromo or 5-fluoro), 5-trifluoromethyl, or other 5-substituted uracilsand cytosines; and 6-azouracil. Further useful nucleotide bases can befound in Kurreck, 2003 Eur. J. Biochem. 270:1628; Herdewijn, 2000 GuideNucleic Acid Develop. 10:297; Concise Encyclopedia of Polymer Scienceand Engineering, pp. 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990; U.S. Pat. No. 3,687,808, and similar references, all of which areincorporated by reference herein.

Certain modified nucleotide base moieties are also contemplated. Theseinclude 5-substituted pyrimidines; 6-azapyrimidines; and N-2, N-6, orO-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine). Further, for example, 5-methyluridine and5-methylcytosine substitutions are known to increase nucleic acid duplexstability, which can be combined with 2′-sugar modifications (e.g.,2′-O-methyl or 2′-methoxyethyl) or internucleoside linkages (e.g.,phosphorothioate) that provide the desired nuclease resistance to themodified or substituted segmented miRNA mimetics.

In certain embodiments, at least one pyrimidine of a segmented miRNAmimetic of the invention is a locked nucleic acid (LNA) in the form of abicyclic sugar. In a related embodiment, the LNA comprises a basesubstitution, such as a 5-methyluridine LNA or 2-thio-5-methyluridineLNA. In certain embodiments, a ribose of the pyrimidine nucleoside orthe internucleoside linkage can be optionally modified.

In any of these embodiments, one or more modified nucleotides can be a Gclamp (e.g., a cytosine analog that forms an additional hydrogen bond toguanine, such as 9-(aminoethoxy) phenoxazine). See, e.g., Lin andMateucci, 1998 Nucleic Acids Res. 19:3111).

In addition, the terminal structure of segmented miRNA mimetics of thisdisclosure can comprise a stem-loop structure in which an end of onestrand (e.g., the guide strand) of a segmented miRNA mimetic isconnected by a linker nucleic acid, e.g., a linker RNA to an end of theopposite strand (e.g., the passenger strand). When linker segments areemployed, there is no particular limitation in the length of the linkeras long as it does not hinder pairing of the stem portion. For example,for stable pairing of the stem portion, the linker portion can have aclover-leaf tRNA structure. Even if the linker has a length that wouldhinder pairing of the stem portion, it is possible, for example, toconstruct the linker portion to include introns so that the introns areexcised during processing of a precursor miRNA mimetic into a maturemiRNA mimetic, thereby allowing pairing of the stem portion. In the caseof a stem-loop dsRNA, either end (head or tail) of a segmented miRNAmimetic with no loop structure can comprise a low molecular weight RNA,for example, a natural RNA molecule such as a tRNA, rRNA or viral RNA,or an artificial RNA molecule.

A segmented miRNA mimetic of the invention can be constructed such thatit takes on an overall circular structure, wherein the entire moleculeis about 10 to about 60 nucleotides in length having from about 5 toabout 26 base pairs (e.g., about 19 to about 21) wherein the circularoligonucleotide forms a dumbbell shaped structure having about 10 toabout 26 base pairs and two loops. In certain embodiments, a circularsegmented miRNA mimetic contains two loop motifs, wherein one or bothloop portions are biodegradable.

In another aspect of this disclosure, the segmented miRNA mimeticstructures of the invention and their potential of allowing moresuitable types of chemical modification and allowing modification to ahigher extent can be used to reduce interferon activation when asegmented miRNA mimetic is contacted with a biological sample, forexample, when it is introduced into a eukaryotic cell. A segmented miRNAmimetic of the invention comprises at least 6 ends, including terminaland internal ends, as compared to its traditional non-segmented duplexmiRNA mimetic counterpart, which comprises 4 ends. These ends canconveniently be used for tethering functional chemical groups toenhance, for example, lipophilic and other properties associated withcellular delivery. For instance, it is possible to tether bulky groupslike cholesterol to the 5′-ends of each of the contiguous stretches ofnucleotides without losing RNAi activity.

Moreover, because the yield of synthesis is usually higher for shorterRNA strands, the cost of large-scale synthesis in connection withtherapeutic application can be substantially reduced using the segmentedmiRNA mimetics of the present invention.

In any of the embodiments described herein, a segmented miRNA mimeticcan include multiple types of modifications in combination. For example,a segmented miRNA mimetic having at least one ribothymidine or2-thioribothymidine can further comprise at least one LNA, 2′-methoxy,2′-fluoro, 2′-deoxy, phosphorothioate linkage, an inverted base terminalcap, or any combination thereof. In certain exemplary embodiments, asegmented miRNA mimetic can comprise one or more or all uridines (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substituted with 2′-O-methyluridine and have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LNAsubstitutions. In other exemplary embodiments, a segmented miRNA mimeticcan comprise from one or more or all uridines substituted with2′-O-methyl uridine and have up to about 25% phosphorothioatesubstitutions. In still other exemplary embodiments, a segmented miRNAmimetic can comprise one or more or all uridines (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more) substituted with 2′-O-methyl uridine and have 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2′-deoxy-2′-fluoro substitutions.

Within certain aspects, the present disclosure also provides segmentedmiRNA molecules comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more) universal base nucleotides. In certain aspects, a segmentedmiRNA mimetic disclosed herein can include about 1 to about 10 universalbase nucleotides, so long as the resulting segmented miRNA mimeticremains capable of modulating one or more of its endogenous miRNAtargets.

Suitable modifications can also include one or more suitable conjugatesattached to, typically the ends, including the terminal ends andinternal ends, of a segmented miRNA mimetic of the invention. Theconjugate can be attached via a covalent attachment. In some cases, theconjugate can be linked to the segmented miRNA mimetic via abiodegradable linker, attached the 3′-end, 5′ end, or both ends of thepassenger strand, the guide strand and/or the internal ends of each ofthe contiguous stretches of nucleotides. The conjugate molecule canfacilitate the delivery of the double-stranded oligonucleotide moleculeinto a biological system, such as a cell. The conjugate can also be apolyethylene glycol (PEG), human serum albumin, or a ligand for acellular receptor that can facilitate cellular uptake. However, asexplained above, the endogenous miRNA and siRNA paths of biogenesis andmachineries are distinct, featuring different components orparticipants, therefore conjugates or other modifications in this classthat are suitable for an exogenously introduced siRNA molecule can stillbe unsuitable for an exogenously introduced miRNA mimetic molecule suchas a segmented miRNA mimetic of the invention.

Substitutions and Insertions

Various non-nucleotide moieties as are provided herein or otherwiseknown in the art can be used as substitutions and/or insertions in thesegmented miRNA mimetics of the invention provided that RNAi activityagainst one or more miRNA targets is maintained.

In one aspect of the invention, substitutions and/or insertions in thesegmented mimetic miRNAs of the invention can comprise one or more alkylmoieties, e.g., any C₁-C₂₀, and preferably a C₁, C₂, C₃, C₄, C₅, C₆, C₇,C₈, C₉ or C₁₀ alykl moiety. The alykl moiety can be straight chain,branched, aliphatic or aromatic. The alkyl moiety can be substituted orunsubstituted. In certain embodiments the alkyl moieties are C₃ and/orC₆.

Segmented mimetic miRNAs of the present invention can comprisesubstitutions or insertions that incorporate one or more smallmolecules, lipids or lipophiles, terpenes, phospholipids, antibodies,toxins, cholesterol, a protein binding agent (e.g., a ligand for acellular receptor that can facilitate cellular uptake), a vitamin,negatively charged polymers and other polymers, for example proteins(e.g., human serum albumin), peptides, hormones, carbohydrates,polyethylene glycols, or polyamines, and those described in, forexample, U.S. Patent Publication No. 2005/0196781, and U.S. PatentPublication No. 2006/0293271, the disclosures of which are incorporatedherein by reference. Substitutions and insertions can include alkylchains optionally substituted with a functional group. For example, thealkyl chain can be substituted with a moiety that binds specifically toa target molecule of interest.

Substitutions and insertions of the present invention can furthercomprise a polyether, polyamine, polyamide, peptide, carbohydrate,lipid, polyhydrocarbon, or other polymeric compounds (e.g., polyethyleneglycols such as those having between 2 and 100 ethylene glycol units).Specific examples include those described by Seela and Kaiser, 1990,Nucleic Acids Res. 18:6353; Seela and Kaiser, 1987, Nucleic Acids Res.15:3113; Cload and Schepartz, 1991, J. Am. Chem. Soc. 113:6324;Richardson and Schepartz, 1991, J. Am. Chem. Soc. 113:5109; Ma et al.,1993, Nucleic Acids Res. 27:2585; Ma et al., 1993, Biochemistry 32:1751;Durand et al., 1990, Nucleic Acids Res. 18:6353; McCurdy et al., 1991,Nucleosides & Nucleotides 70:287; Jaschke et al., 1993, TetrahedronLett. 34:301; Ono et al., 1991, Biochemistry 30:9914; and others. Achemical moiety that provides additional functionality (e.g.,specifically binds to a target molecule of interest orfacilitates/enhances cellular delivery of the molecule) to the miRNAmimetic can be a part of the substitution or insertion or covalentlyattached or linked thereto. For example, the additional functional groupcan impart therapeutic activity to miRNA mimetic of the invention byassisting in transferring the RNAi molecule compounds across cellularmembranes, altering the pharmacokinetics, and/or modulating thelocalization of RNAi molecules of the invention.

Substitutions and insertions of the present invention can aid indelivery and/or localization of RNAi molecules of the invention into anumber of cell types originating from different tissues, in the presenceor absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038).For example, the conjugate member can be naproxen, nitroindole (oranother conjugate that contributes to stacking interactions), folate,ibuprofen, or a C5 pyrimidine linker. The conjugate member can be aglyceride lipid conjugate (e.g., a dialkyl glyceride derivatives),vitamin E conjugates, or thio-cholesterols. The conjugate molecule canalternatively be a peptide that functions, when conjugated to a miRNAmimetic, to facilitate delivery of the molecule into a target cell, orotherwise enhance delivery, stability, or activity of the molecule whencontacted with a biological sample. Exemplary peptide conjugate membersfor use within these aspects of this disclosure, include peptides PN27,PN28, PN29, PN58, PN61, PN73, PN158, PN159, PN173, PN182, PN202, PN204,PN250, PN361, PN365, PN404, PN453, and PN509 as described, for example,in U.S. Patent Application Publication Nos. 2006/0040882 and2006/0014289, and U.S. Provisional Patent Application No. 60/939,578,which are all incorporated herein by reference.

A substitution or insertion can comprise a moiety that specificallybinds to a target molecule. The target molecule can be any molecule ofinterest. For example, the target molecule can be a ligand-bindingdomain of a protein, thereby preventing or competing with theinteraction of the naturally-occurring ligand with the protein. This isa non-limiting example and those in the art will recognize that otherembodiments can be readily generated using techniques generally known inthe art (see, e.g., Gold et al, 1995, Annu. Rev. Biochem. 64:163; Brodyand Gold, 2000, J. Biotechnol. 74:5; Sun, 2000, Curr. Opin. Mol. Ther.2:100; Kusser, J., 2000, Biotechnol. 74:21; Hermann and Patel, 2000,Science 257:820; and Jayasena, 1999, Clinical Chem. 45:1628).

The substitution or insertion can provide the ability to administer thesegmented miRNA mimetic to specific cell types, such as hepatocytes. Forexample, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J.Biol. Chem. 262:4429) is unique to hepatocytes and binds branchedgalactose-terminal glycoproteins, such as asialoorosomucoid (ASOR).Binding of such glycoproteins or synthetic glycoconjugates to thereceptor takes place with an affinity that strongly depends on thedegree of branching of the oligosaccharide chain, for example,triatennary structures are bound with greater affinity than biatenarryor monoatennary chains (Baenziger and Fiete, 1980, Cell 22: 611;Connolly et al., 1982, J. Biol. Chem. 257:939). Lee and Lee (1987,Glycoconjugate J. 4:317) obtained this high specificity through the useof N-acetyl-D-galactosamine as the carbohydrate moiety, which has higheraffinity for the receptor compared to galactose. This “clusteringeffect” has also been described for the binding and uptake ofmannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al.,1981, J. Med. Chem. 24: 1388). The use of galactose and galactosaminebased conjugates to transport exogenous compounds across cell membranescan provide a targeted delivery approach to the treatment of liverdisease. The use of bioconjugates can also provide a reduction in therequired dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use ofbioconjugates of this disclosure.

Terminal Analogs, Modifications, Linkers, and Conjugates

In one embodiment, a miRNA mimetic of the invention comprises one ormore terminal nucleotide analogs, non-nucleotide analogs, nucleotidelinkers, non-nucleotide linkers, caps, conjugates and the like as aregenerally known in the art, at the 5′-end, 3′-end, or both 5′- and3′-ends of the passenger strand, or alternately at the 3′-end of theguide strand.

In certain embodiments, the invention features a nucleic acid linkerthat covalently attaches on strand to the other. A nucleotide linker canbe a nucleic acid aptamer. A non-nucleotide linker can be an abasicnucleotide, polyether, polyamine, polyamide, peptide, carbohydrate,lipid, polyhydrocarbon, or other polymeric compounds (e.g., polyethyleneglycols such as those having between 2 and 100 ethylene glycol units).Specific examples include those described by Seela and Kaiser, 1990Nucleic Acids Res. 18:6353; Seela and Kaiser, 1987 Nucleic Acids Res.15:3113; Cload and Schepartz, 1991 J. Am. Chem. Soc. 113:6324;Richardson and Schepartz, 1991 J. Am. Chem. Soc. 113:5109; Ma et al.,1993 Nucleic Acids Res. 27:2585; Ma et al., 1993 Biochemistry 32:1751;Durand et al., 1990 Nucleic Acids Res. 18:6353; McCurdy et al., 1991Nucleosides & Nucleotides 70:287; Jaschke et al., 1993 Tetrahedron Lett.34:301; Ono et al., 1991 Biochemistry 30:9914; and others.

In another embodiment, a conjugate molecule can be optionally attachedto a segmented miRNA mimetic or an analog thereof. For example, suchconjugate molecules can be polyethylene glycol, human serum albumin, ora ligand for a cellular receptor that can, for example, mediate cellularuptake. The conjugate molecule can be attached at one or more of theterminal ends and/or one or more of the internal ends. Examples ofspecific conjugate molecules contemplated by the instant disclosure aredescribed in, for example, U.S. Patent Publication No. 2005/0196781 A1,and U.S. Patent Publication No. 2006/0293271 A1, the disclosures ofwhich are incorporated herein by reference.

In a certain aspect, the invention features conjugates and/or complexesof segmented miRNA mimetics of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of one or more segmentedmiRNA mimetics to a biological system, such as a cell. The conjugatesand complexes provided by the instant invention can impart therapeuticactivity by transferring therapeutic compounds across cellularmembranes, altering the pharmacokinetics, and/or modulating thelocalization of nucleic acid molecules of the invention. The presentinvention encompasses the design and synthesis of conjugates andcomplexes for the delivery of molecules, including, but not limited to,small molecules, lipids, phospholipids, nucleosides, nucleotides,nucleic acids, antibodies, toxins, negatively charged polymers and otherpolymers, for example proteins, peptides, hormones, carbohydrates,polyethylene glycols, or polyamines, across cellular membranes. Ingeneral, the transporters described are designed to be used eitherindividually or as part of a multi-component system, with or withoutdegradable linkers. These compounds are expected to improve deliveryand/or localization of segmented miRNA mimetics of the invention into anumber of cell types originating from different tissues, in the presenceor absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038).Conjugates described herein can be attached to biologically activesegmented miRNA mimetics via linkers that are biodegradable, such asbiodegradable nucleic acid linker molecules.

A person of skill in the art can screen segmented miRNA mimetics of thisdisclosure having various conjugates to determine which of the segmentedmiRNA-conjugate complexes possess improved properties (e.g.,pharmacokinetic profile, bioavailability, stability) while maintainingthe ability to mediate RNAi in, for example, an animal model asdescribed herein or generally known in the art.

In another aspect, a segmented miRNA mimetic of the invention comprisesone or more 5′- and/or a 3′-cap structures, for example at the terminalends of the passenger strand, guide strand, both strands, or any of theinternal ends of the contiguous stretches of nucleotides. Innon-limiting examples: a suitable 5′-cap can be one selected from thegroup comprising inverted abasic residue (moiety); LNA; 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide;carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety.

In another non-limiting example, a suitable 3′-cap can be selected froma group comprising, LNA; 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate;3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties. For more details, seeBeaucage and Iyer, 1993, Tetrahedron 49:1925, which is incorporated byreference herein.

Making microRNA Mimetics of the Invention

Exemplary molecules of the instant disclosure can be recombinantlyproduced (e.g., isolated), chemically synthesized, or a combinationthereof. Oligonucleotides or individual contiguous stretches ofnucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example, as described in Caruthers etal., 1992 Methods in Enzymol. 211:3; Thompson et al, PCT Publication No.WO 99/54459, Wincott et al., 1995 Nucleic Acids Res. 23:2677; Wincott etal., 1997 Methods Mol. Bio. 74:59; Brennan et al., 1998 BiotechnohBioeng. 67:33; and Brennan, U.S. Pat. No. 6,001,311. Synthesis of RNA,including certain segmented miRNA mimetics thereof of this disclosure,can be made using the procedure as described in Usman et al., 1987 J.Am. Chem. Soc. 109:7845; Scaringe et al., 1990 Nucleic Acids Res.18:5433; and Wincott et al., 1995 Nucleic Acids Res. 23:2677; Wincott etal., 1997 Methods Mol. Bio. 74:59. In certain embodiments, segmentedmiRNA mimetics of the present disclosure can be synthesized separatelyand joined together post-synthetically, for example, by ligation (Mooreet al., 1992 Science 256:9923; Draper et al., PCT Publication No. WO93/23569; Shabarova et al., 1991 Nucleic Acids Res. 19:4247; Bellon etal., 1997 Nucleosides & Nucleotides 16:951; Bellon et al., 1997Bioconjugate Chem. 8:204), or by hybridization following synthesis ordeprotection. In certain embodiments, a segmented miRNA mimetic of thisdisclosure can be made as single or multiple transcription productsexpressed by a polynucleotide vector encoding one or more contiguousstretches of RNAs and directing their expression within host cells. Inall of the embodiments herein, the double-stranded portion of a finaltranscription product to be expressed within the target cell can be, forexample, about 10 to about 26 bp, about 12 to about 25 bp, or about 14to about 22 bp long.

Methods for Designing a Segmented miRNA Mimetic

As described herein, a segmented miRNA mimetic can be designed based ona corresponding non-segmented duplex miRNA mimetic molecule, which is inturn designed based on known endogenous miRNA molecules, such as thoselisted in the miRBase as of the filing date of the present application,and for example, SEQ ID Nos: 1-1090 in Table I. The segmented miRNAmimetic is then characterized as described below and in the Examplesherein.

Specifically, any segmented miRNA mimetic of the invention can bedesigned by introducing one or more discontinuities of the invention(nicks, gaps, substitutions, and/or insertions) into the passengerstrand, the guide strand, or both the passenger and the guide strands.The discontinuity can be introduced at the 5′-end of any of position 2,position 3, position 4, position 5, position 6, position 7, position 8,position 9, position 10, position 11, position 12, position 13, position14, position 15, position 16, position 17, position 18, position 19,position 20, position 21, position 22, position 23, position 24,position 25, and/or position 26 of the guide strand. The discontinuitycan be introduced at the 5′-end of any of position 2, position 3,position 4, position 5, position 6, position 7, position 8, position 9,position 10, position 11, position 12, position 13, position 14,position 15, position 16, position 17, position 18, position 19,position 20, position 21, position 22, position 23, position 24,position 25, and/or position 26 of the passenger strand.

In certain embodiments, a method is provided wherein one or more genes,which are known to be regulated by an endogenous miRNA, are selected toindicate the RNAi potency of a segmented miRNA mimetic. The RNAiactivity of a given segmented miRNA mimetic can be measured using knownmethods, such as those described generally in Fire et al., PCTPublication No. WO99/32619. In some embodiments, the instantspecification provides methods for selecting more efficacious segmentedmiRNA mimetic designs by using one or more reporter gene constructscomprising a constitutive promoter, such as a cytomegalovirus (CMV) orphosphoglycerate kinase (PGK) promoter, operably fused to, and capableof altering the expression of one or more reporter genes, such as aluciferase, chloramphenicol (CAT), or β-galactosidase, which, in turn,is operably fused in-frame with a segmented miRNA mimetic. Thesereporter gene expression constructs can be co-transfected with one ormore segmented miRNA mimetics and the control (corresponding)non-segmented miRNA mimetics. The capacity of a given segmented miRNAmimetic to mediate RNAi of a target mRNA can be determined by comparingthe measured reporter gene activity in cells transfected with thesegmented miRNA mimetic and the activity in cells transfected with anegative control (i.e., in cells not transfected with the segmentedmiRNA mimetic) and a positive control (i.e., in cells transfected withthe corresponding non-segmented duplex miRNA mimetic). The segmentedmiRNA mimetics having at least 20% or more, preferably at least 40% ormore, or 60% or more, or 80% or more, of the activity of theircorresponding non-segmented duplex miRNA mimetics are selected.

Certain embodiments disclosed herein also provide methods for selectingone or more segmented miRNA mimetics based on their predicted stability.A theoretical melting curve can be prepared for each of the segmentedmiRNA mimetic designs such that those with high theoretical meltingcurves, and therefore higher duplex stability and corresponding lowercytotoxic effects, would be selected. Alternatively, stability of asegmented miRNA mimetic can be determined empirically and those withhigher measured melting temperatures would be selected.

Compositions and Methods of Use

As set forth herein, segmented miRNA mimetics of the invention are miRNAmimetics that are designed to supplement or take the place ofcorresponding naturally-occurring miRNAs, the reduced or otherwiseunsuitably low levels of which have been associated with pathological ordiseased conditions. A segmented miRNA mimetic of the invention istherefore preferably capable of participating in the cellular RNAipathway or otherwise capable of modulating the same or relatedpathway(s). A segmented miRNA mimetic of the invention can be introducedto a cell, tissue, organism, an in vitro, or an in vivo system tomediate RNAi against an endogenous RNA target of its correspondingnaturally-occurring miRNA. As such, a segmented miRNA mimetic canregulate a number of genes, for example, downstream from its RNA target,whose expression levels are associated with or otherwise regulated bythe corresponding naturally-occurring miRNA. Because aberrant expressionlevels of certain naturally-occurring miRNAs have been implicated invarious human ailments, including, but are not limited to,hyperproliferative, angiogenic, or inflammatory diseases, states, oradverse conditions, the segmented miRNA mimetics of the presentinvention can offer valuable therapeutic opportunities. In this context,a segmented miRNA mimetic of this disclosure can regulate (e.g.,knockdown or up-regulate) expression of one or more downstream genes ofits corresponding endogenous miRNA, such that prevention, alleviation,or reduction of the severity or recurrence of one or more associateddisease symptoms can be achieved. Alternatively, for various distinctdisease models in which expression of one or more target mRNAs are notnecessarily reduced or at a lower-than-normal level as a consequence orsequel of diseases or other adverse conditions, introducing exogenousmiRNA mimetics, such as one or more segmented miRNA mimetics of theinvention, can nonetheless result in a therapeutic result by affectingthe expression levels of genes associated with the disease pathway. Thesegmented miRNA mimetics of the invention thus are useful reagents,which can be in methods for a variety of therapeutic, diagnostic, targetvalidation, genomic discovery, genetic engineering, and pharmacogenomicapplications.

In certain embodiments, aqueous suspensions containing one or moresegmented miRNA mimetics of the invention can be prepared in admixturewith suitable excipients, such as suspending agents or dispersing orwetting agents. Exemplary suspending agents include sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.Representative dispersing or wetting agents include naturally-occurringphosphatides (e.g., lecithin), condensation products of an alkyleneoxide with fatty acids (e.g., polyoxyethylene stearate), condensationproducts of ethylene oxide with long chain aliphatic alcohols (e.g.,heptadecaethyleneoxycetanol), condensation products of ethylene oxidewith partial esters derived from fatty acids and hexitol (e.g.,polyoxyethylene sorbitol monooleate), or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides (e.g., polyethylene sorbitan monooleate). In certainembodiments, the aqueous suspensions can optionally contain one or morepreservatives (e.g., ethyl or w-propyl-p-hydroxybenzoate), one or morecoloring agents, one or more flavoring agents, or one or more sweeteningagents (e.g., sucrose, saccharin). In additional embodiments,dispersible powders and granules suitable for preparation of an aqueoussuspension comprising one or more segmented miRNA mimetics of theinvention can be prepared by the addition of water with the segmentedmiRNA mimetics in admixture with a dispersing or wetting agent,suspending agent and optionally one or more preservative, coloringagent, flavoring agent, or sweetening agent. The present disclosure alsoincludes segmented miRNA mimetic compositions prepared for storage oradministration that include a pharmaceutically effective amount of adesired compound in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co., A. R. Gennaro edit., 21stEdition, 2005. In certain embodiments, pharmaceutical compositions ofthis disclosure can optionally include preservatives, antioxidants,stabilizers, dyes, flavoring agents, or any combination thereof.Exemplary preservatives include sodium benzoate, esters ofp-hydroxybenzoic acid, and sorbic acid.

The segmented miRNA mimetic compositions of the instant disclosure canbe effectively employed as pharmaceutically-acceptable formulations.Pharmaceutically-acceptable formulations prevent, alter the occurrenceor severity of, or treat (alleviate one or more symptom(s) to adetectable or measurable extent) a disease state or other adversecondition in a subject. A pharmaceutically acceptable formulationincludes salts of the above compounds, for example, acid addition salts,such as salts of hydrochloric acid, hydrobromic acid, acetic acid, orbenzene sulfonic acid. A pharmaceutical composition or formulationrefers to a composition or formulation in a form suitable foradministration into a cell, or a subject such as a human (e.g., systemicadministration). The formulations of the present disclosure, having anamount of segmented miRNA mimetic sufficient to treat or prevent adisorder associated with target gene expression are, for example,suitable for topical (e.g., creams, ointments, skin patches, eye drops,ear drops) application or administration. Other routes of administrationinclude oral, parenteral, sublingual, bladder washout, vaginal, rectal,enteric, suppository, nasal, and inhalation. The pharmaceuticalcompositions of the present disclosure are formulated to allow thesegmented miRNA mimetic contained therein to be bioavailable uponadministration to a subject.

In certain embodiments, a segmented miRNA of this disclosure can beformulated as oily suspensions or emulsions (e.g., oil-in-water) bysuspending the segmented miRNA mimetic in, for example, a vegetable oil(e.g., arachis oil, olive oil, sesame oil or coconut oil) or a mineraloil (e.g., liquid paraffin). Suitable emulsifying agents can benaturally-occurring gums (e.g., gum acacia or gum tragacanth),naturally-occurring phosphatides (e.g., soy bean, lecithin, esters orpartial esters derived from fatty acids and hexitol), anhydrides (e.g.,sorbitan monooleate), or condensation products of partial esters withethylene oxide (e.g., polyoxyethylene sorbitan monooleate). In certainembodiments, the oily suspensions or emulsions can optionally contain athickening agent, such as beeswax, hard paraffin or cetyl alcohol. Inrelated embodiments, sweetening agents and flavoring agents canoptionally be added to provide palatable oral preparations. In yet otherembodiments, these compositions can be preserved by the optionallyadding an anti-oxidant, such as ascorbic acid.

In certain embodiments, a segmented miRNA mimetic can be formulated assyrups and elixirs with sweetening agents (e.g., glycerol, propyleneglycol, sorbitol, glucose or sucrose). Such formulations can alsocontain a demulcent, preservative, flavoring, coloring agent, or anycombination thereof. In other embodiments, pharmaceutical compositionscomprising a segmented miRNA mimetic can be in the form of a sterile,injectable aqueous or oleaginous suspension. The sterile injectablepreparation can also be a sterile, injectable solution or suspension ina non-toxic parenterally acceptable diluent or solvent (e.g., as asolution in 1,3-butanediol). Among the exemplary acceptable vehicles andsolvents useful in the compositions of this disclosure is water,Ringer's solution, or isotonic sodium chloride solution. In addition,sterile, fixed oils can be employed as a solvent or suspending medium.For this purpose, any bland fixed oil can be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of parenteral formulations.

Within certain embodiments of this disclosure, pharmaceuticalcompositions and methods are provided that feature the presence oradministration of one or more segmented miRNA mimetics, combined,complexed, or conjugated with a polypeptide, optionally formulated witha pharmaceutically-acceptable carrier, such as a diluent, stabilizer,buffer, or the like. The negatively charged segmented miRNA mimetics canbe administered to a patient by any standard means, with or withoutstabilizers, buffers, or the like, to form a composition suitable fortreatment. When it is desired to use a liposome delivery mechanism,standard protocols for formation of liposomes can be followed. Thecompositions of the present disclosure can also be formulated and usedas a tablet, capsule or elixir for oral administration, suppository forrectal administration, sterile solution, or suspension for injectableadministration, either with or without other compounds known in the art.Thus, a segmented miRNA mimetic of the present disclosure can beadministered in any form, such as nasally, transdermally, parenterally,or by local injection.

In accordance with this disclosure herein, a segmented miRNA mimetic(optionally substituted or modified or conjugated), compositionsthereof, and methods for inhibiting expression of one or morecorresponding target mRNAs in a cell or organism are provided. Incertain embodiments, this disclosure provides methods and segmentedmiRNA mimetic compositions for treating a subject, including a humancell, tissue or individual, having a disease or at risk of developing adisease caused by or associated with the aberrant levels of itscorresponding naturally-occurring miRNA. In a certain embodiment, themethod includes administering a segmented miRNA mimetic or apharmaceutical composition containing the segmented miRNA mimetic to acell or an organism, such as a mammal, such that the level of itscorresponding naturally-occurring miRNA within the cell or the organismis increased. Subjects (e.g., mammalian, human) amendable for treatmentusing the segmented miRNA mimetics (optionally substituted or modifiedor conjugated), compositions thereof, and methods of the presentdisclosure include those suffering from one or more disease or conditionmediated, at least in part, by an aberrant expression level of itscorresponding naturally-occurring miRNA, or which are amenable totreatment by replenishing or increasing the level of RNAi mediated bythe corresponding miRNA, including a hyperproliferative (e.g., cancer),angiogenic, metabolic, or inflammatory (e.g., arthritis) disease ordisorder or condition.

Compositions and methods disclosed herein are useful in the treatment ofa wide variety of target viruses, including retrovirus, such as humanimmunodeficiency virus (HIV), Hepatitis C Virus, Hepatitis B Virus,Coronavirus, as well as respiratory viruses, including human RespiratorySyncytial Virus, human Metapneumovirus, human Parainfluenza virusRhinovirus and Influenza virus.

In other examples, the compositions and methods of this disclosure areuseful as therapeutic tools to treat or prevent symptoms of, forexample, hyperproliferative disorders. Exemplary hyperproliferativedisorders include neoplasms, carcinomas, sarcomas, tumors, or cancer.More exemplary hyperproliferative disorders include oral cancer, throatcancer, laryngeal cancer, esophageal cancer, pharyngeal cancer,nasopharyngeal cancer, oropharyngeal cancer, gastrointestinal tractcancer, gastrointestinal stromal tumors (GIST), small intestine cancer,colon cancer, rectal cancer, colorectal cancer, anal cancer, pancreaticcancer, breast cancer, cervical cancer, uterine cancer, vulvar cancer,vaginal cancer, urinary tract cancer, bladder cancer, kidney cancer,adrenocortical cancer, islet cell carcinoma, gallbladder cancer, stomachcancer, prostate cancer, ovarian cancer, endometrial cancer,trophoblastic tumor, testicular cancer, penial cancer, bone cancer,osteosarcoma, liver cancer, extrahepatic bile duct cancer, skin cancer,basal cell carcinoma (BCC), lung cancer, small cell lung cancer,non-small cell lung cancer (NSCLC), brain cancer, melanoma, Kaposi'ssarcoma, eye cancer, head and neck cancer, squamous cell carcinoma ofhead and neck, tymoma, thymic carcinoma, thyroid cancer, parathyroidcancer, Hippel-Lindau syndrome, leukemia, acute myeloid leukemia,chronic myelogenous leukemia, acute lymphoblastic leukemia, hairy cellleukemia, lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, T-celllymphoma, multiple myeloma, malignant pleural mesothelioma, Barrett'sadenocarcinoma, Wilm's tumor, or the like. In other examples, thecompositions and methods of this disclosure are useful as therapeutictools to regulate expression of one or more target gene to treat orprevent symptoms of, for example, inflammatory disorders. Exemplaryinflammatory disorders include diabetes mellitus, rheumatoid arthritis,pannus growth in inflamed synovial lining, collagen-induced arthritis,spondylarthritis, ankylosing spondylitis, multiple sclerosis,encephalomyelitis, inflammatory bowel disease, Chron's disease,psoriasis or psoriatic arthritis, myasthenia gravis, systemic lupuserythematosis, graft-versus-host disease, atherosclerosis, andallergies.

Other exemplary disorders that can be treated with segmented miRNAmimetics, compositions and methods of the instant disclosure includemetabolic disorders, cardiac disease, pulmonary disease,neovascularization, ischemic disorders, age-related maculardegeneration, diabetic retinopathy, glomerulonephritis, diabetes,asthma, chronic obstructive pulmonary disease, chronic bronchitis,lymphangiogenesis, and atherosclerosis.

Within additional aspects, combination formulations and methods areprovided comprising an effective amount of one or more segmented miRNAmimetics in combination with one or more secondary or adjunctive activeagents that are formulated together or administered coordinately withthe segmented miRNA mimetics of the invention to control one or moretarget gene-associated disease or condition as described herein. Usefuladjunctive therapeutic agents in these combinatorial formulations andcoordinate treatment methods include, for example, enzymatic nucleicacid molecules, allosteric nucleic acid molecules, guide, decoy, oraptamer nucleic acid molecules, antibodies such as monoclonalantibodies, small molecules and other organic or inorganic compoundsincluding metals, salts and ions, and other drugs and active agentsindicated for treating one or more target gene-associated disease orcondition, including chemotherapeutic agents used to treat cancer,steroids, non-steroidal anti-inflammatory drugs (NSAIDs), or the like.Exemplary chemotherapeutic agents include alkylating agents (e.g.,cisplatin, oxaliplatin, carboplatin, busulfan, nitrosoureas, nitrogenmustards, uramustine, temozolomide), antimetabolites (e.g., aminopterin,methotrexate, mercaptopurine, fluorouracil, cytarabine), taxanes (e.g.,paclitaxel, docetaxel), anthracyclines (e.g., doxorubicin, daunorubicin,epirubicin, idaruicin, mitoxantrone, valrubicin), bleomycin, mytomycin,actinomycin, hydroxyurea, topoisomerase inhibitors (e.g., camptothecin,topotecan, irinotecan, etoposide, teniposide), monoclonal antibodies(e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab, panitumumab,rituximab, tositumomab, trastuzumab), vinca alkaloids (e.g.,vincristine, vinblastine, vindesine, vinorelbine), cyclophosphamide,prednisone, leucovorin, oxaliplatin. To practice the coordinateadministration methods of this disclosure, a segmented miRNA mimetic isadministered simultaneously or sequentially in a coordinated treatmentprotocol with one or more secondary or adjunctive therapeutic agentsdescribed herein or known in the art. The coordinate administration canbe done in either order, and there can be a time period while only oneor both (or all) active therapeutic agents, individually orcollectively, exert their biological activities. A distinguishing aspectof all such coordinate treatment methods is that the segmented miRNAmimetic present in a composition elicits some favorable clinicalresponse, which can or can not be in conjunction with a secondaryclinical response provided by the secondary therapeutic agent. Forexample, the coordinate administration of a segmented miRNA mimetic witha secondary therapeutic agent as contemplated herein can yield anenhanced (e.g., synergistic) therapeutic response beyond the therapeuticresponse elicited by either or both the purified segmented miRNA mimeticand the secondary therapeutic agent alone.

In another embodiment, a segmented miRNA mimetic of this disclosure caninclude a conjugate member on one or more of the nucleotides, at theterminal positions or the internal positions. The conjugate member canbe, for example, a lipophile, a terpene, a protein binding agent, avitamin, a carbohydrate, or a peptide. For example, the conjugate membercan be naproxen, nitroindole (or another conjugate that contributes tostacking interactions), folate, ibuprofen, or a C5 pyrimidine linker. Inother embodiments, the conjugate member is a glyceride lipid conjugate(e.g., a dialkyl glyceride derivatives), vitamin E conjugates, orthio-cholesterols. Additional conjugate members include peptides thatfunction, when conjugated to a modified segmented miRNA mimetic, tofacilitate delivery of the mimetic into a target cell, or otherwiseenhance delivery, stability, or activity of the mimetic when contactedwith a biological sample. Exemplary peptide conjugate members for usewithin these aspects of this disclosure, include peptides PN27, PN28,PN29, PN58, PN61, PN73, PN158, PN159, PN173, PN182, PN202, PN204, PN250,PN361, PN365, PN404, PN453, and PN509 are described, for example, inU.S. Patent Application Publication Nos. 2006/0040882 and 2006/0014289,and U.S. Provisional Patent Application No. 60/939,578, which are allincorporated herein by reference. In certain embodiments, when peptideconjugate partners are used to enhance delivery of one or more segmentedmiRNA mimetics of this disclosure, the resulting formulations andmethods will often exhibit further reduction of an interferon responsein target cells as compared to a segmented miRNA mimetic delivered incombination with alternate delivery vehicles, such as lipid deliveryvehicles (e.g., Lipofectamine™). In still another embodiment, asegmented miRNA mimetic of the invention can be conjugated to apolypeptide and admixed with one or more non-cationic lipids or acombination of a non-cationic lipid and a cationic lipid to form acomposition that enhances intracellular delivery of the segmented miRNAmimetic as compared to delivery resulting from contacting the targetcells with a naked segmented miRNA mimetic without the lipids. In moredetailed aspects of this disclosure, the mixture, complex or conjugatecomprising a segmented miRNA mimetic and a polypeptide can be optionallycombined with (e.g., admixed or complexed with) a cationic lipid, suchas Lipofectine™. To produce these compositions comprised of apolypeptide, a segmented miRNA mimetic and a cationic lipid, thesegmented miRNA mimetic and the polypeptide can be mixed together firstin a suitable medium such as a cell culture medium, after which thecationic lipid is added to the mixture to form an segmented miRNAmimetic/delivery peptide/cationic lipid composition. Optionally, thepeptide and cationic lipid can be mixed together first in a suitablemedium such as a cell culture medium, followed by the addition of thesegmented miRNA mimetic to form the segmented miRNA mimetic/deliverypeptide/cationic lipid composition.

This disclosure also features the use of segmented miRNA mimeticcompositions comprising surface-modified liposomes containingpoly(ethylene glycol) lipids (PEG-modified, or long-circulatingliposomes or stealth liposomes). These formulations can offer increasedaccumulation of drugs in target tissues (Lasic et al., 1995 Chem. Rev.,95:2601; Ishiwata et al., 1995 Chem. Pharm. Bull. 43:1005). Suchliposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., 1995 Science 267:1215; Oku et al., 1995 Biochim.Biophys. Acta 1238:86). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of nucleic acid molecules ascompared to conventional cationic liposomes, which are known toaccumulate in tissues of the mononuclear phagocytic system (MPS) (Liu etal., 1995 J. Biol. Chem. 42:24864; Choi et al., PCT Publication No. WO96/10391; Ansell et al., PCT Publication No. WO 96/10390; Holland etal., PCT Publication No. WO 96/10392). Long-circulating liposomes canalso provide additional protection from nuclease degradation as comparedto cationic liposomes in theory due to avoiding accumulation inmetabolically aggressive MPS tissues, such as the liver and spleen. In acertain embodiment, this disclosure provides compositions suitable foradministering segmented miRNA mimetics of this disclosure to specificcell types, such as hepatocytes. For example, the asialoglycoproteinreceptor (ASGPr) (Wu and Wu, 1987 J. Biol. Chem. 262:4429) is unique tohepatocytes and binds branched galactose-terminal glycoproteins, such asasialoorosomucoid (ASOR). Binding of such glycoproteins or syntheticglycoconjugates to the receptor takes place with an affinity thatstrongly depends on the degree of branching of the oligosaccharidechain, for example, triatennary structures are bound with greateraffinity than biatenarry or monoatennary chains (Baenziger and Fiete,1980 Cell 22: 611; Connolly et al., 1982 J. Biol. Chem. 257:939). Leeand Lee (1987 Glycoconjugate J. 4:317) obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981 J. Med. Chem. 24: 1388). The use of galactose andgalactosamine based conjugates to transport exogenous compounds acrosscell membranes can provide a targeted delivery approach to the treatmentof liver disease. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use ofbioconjugates of this disclosure.

The present disclosure also features a method for preparing segmentedmiRNA mimetic nanoparticles. A first solution containing melaminederivatives is dissolved in an organic solvent such as dimethylsulfoxide, or dimethyl formamide to which an acid such as HCl has beenadded. The concentration of HCl would be about 3.3 moles of HCl forevery mole of the melamine derivative. The first solution is then mixedwith a second solution, which includes a nucleic acid dissolved orsuspended in a polar or hydrophilic solvent (e.g., an aqueous buffersolution containing, for instance, ethylenediaminetraacetic acid (EDTA),or tris(hydroxymethyl) aminomethane (TRIS), or combinations thereof. Themixture forms a first emulsion. The mixing can be done using anystandard technique such as, for example, sonication, vortexing, or in amicro fluidizer. The resultant nucleic acid particles can be purifiedand the organic solvent removed using size-exclusion chromatography ordialysis or both. The complexed nucleic acid nanoparticles can then bemixed with an aqueous solution containing either polyarginine or aGln-Asn polymer, or both, in an aqueous solution. A preferred molecularweight of each polymer is about 5000 to about 15,000 Daltons. This formsa solution containing nanoparticles of nucleic acid complexed with themelamine derivative and the polyarginine and the Gln-Asn polymers. Themixing steps are carried out in a manner that minimizes shearing of thenucleic acid while producing nanoparticles on average smaller than about200 nanometers in diameter. It is believed that the polyargininecomplexes with the negative charge of the phosphate groups within theminor groove of the nucleic acid, and the polyarginine wraps around thetrimeric nucleic acid complex. At either terminus of the polyarginineother moieties, such as the TAT polypeptide, mannose or galactose, canbe covalently bound to the polymer to direct binding of the nucleic acidcomplex to specific tissues, such as to the liver when galactose isused. While not being bound to theory, it is believed that the Gln-Asnpolymer complexes with the nucleic acid complex within the major grooveof the nucleic acid through hydrogen bonding with the bases of thenucleic acid. The polyarginine and the Gln-Asn polymer should be presentat a concentration of 2 moles per every mole of nucleic acid having 20base pairs. The concentration should be increased proportionally for anucleic acid having more than 20 base pairs. For example, if the nucleicacid has 25 base pairs, the concentration of the polymers should be2.5-3 moles per mole of double-stranded nucleic acid. The resultantnanoparticles can be purified by standard means such as size exclusionchromatography followed by dialysis. The purified complexednanoparticles can then be lyophilized using techniques well known in theart. In certain embodiments of the present disclosure providesnanoparticles less than 100 nanometers (nm) comprising a segmented miRNAmimetic.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of subject being treated, the physicalcharacteristics of the specific subject under consideration fortreatment, concurrent medication, and other factors that those skilledin the medical arts will recognize. For example, an amount between 0.1mg/kg and 100 mg/kg body weight/day of active ingredients can beadministered depending on the potency of a segmented miRNA mimetic ofthis disclosure.

Dosage levels in the order of about 0.1 mg to about 140 mg per kilogramof body weight per day can be useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular patientdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy. Following administration of a segmented miRNA mimeticcomposition according to the formulations and methods of thisdisclosure, test subjects will exhibit about a 10% up to about a 99%reduction in one or more symptoms associated with the disease ordisorder being treated, as compared to placebo-treated or other suitablecontrol subjects.

Nucleic acid molecules and polypeptides can be administered to cells ororganisms by a variety of methods known to those of skill in the art,including administration of formulations that comprise a miRNA mimeticand/or a polypeptide alone, or formulations that further comprise one ormore additional components, such as a pharmaceutically acceptablecarrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer,preservative, or the like. In certain embodiments, a segmented miRNAmimetic of the invention, and/or the polypeptide can be encapsulated inliposomes, administered by iontophoresis, or incorporated into othervehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules,bioadhesive microspheres, or proteinaceous vectors (see, e.g., PCTPublication No. WO 00/53722). Alternatively, a nucleicacid/peptide/vehicle combination can be locally delivered by directinjection or by use of an infusion pump. Direct injection of the nucleicacid molecules of this disclosure, whether subcutaneous, intramuscular,or intradermal, can take place using standard needle and syringemethodologies, or by needle-free technologies, such as those describedin Conroy et al, 1999 Clin. Cancer Res. 5:2330; and PCT Publication No.WO 99/31262.

A segmented miRNA mimetic of the invention can also be administered inthe form of suppositories, for example, for rectal administration of thedrug. These compositions can be prepared by mixing the drug with asuitable non-irritating excipient that is solid at ordinary temperaturesbut liquid at the rectal temperature and will therefore melt in therectum to release the drug. Such materials include cocoa butter andpolyethylene glycols.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

Further methods for delivery of nucleic acid molecules, such as asegmented miRNA mimetic of this invention, have been described in, forexample, Boado et al., 1998 J. Pharm. Sci., 87:1308; Tyler et al., 1999FEBS Lett. 421:2m; Pardridge et al., 1995 Proc. Nat'l Acad. Sci. USA92:5592; Boado, 1995 Adv. Drug Delivery Rev. 15:73; Aldrian-Herrada etal. 1998 Nucleic Acids Res. 26:4910; Tyler et al., 1999 Proc. Nat'lAcad. Sci. USA 96:7053; Akhtar et al., 1992 Trends Cell Bio. 2:139;“Delivery Strategies for Guide Oligonucleotide Therapeutics,” ed.Akhtar, 1995, Maurer et al., 1999 Mol Membr. Biol. 16:129; Lee et al.,2000 ACS Symp. Ser., 752:184. In addition to in vivo and therapeuticapplications, a skilled person in the art will appreciate that thesegmented miRNA mimetics of the present disclosure are useful in a widevariety of in vitro applications, such as in scientific and commercialresearch (e.g., elucidation of physiological pathways, drug discoveryand development), and medical and veterinary diagnostics.

All U.S. patents, U.S. patent publications, U.S. patent applications,foreign patents, foreign patent applications, non-patent publications,figures, and websites referred to in this specification are expresslyincorporated herein by reference, in their entirety.

Table I lists certain endogenous mammalian and viral miRNA sequences,wherein the seed sequences, confirmed or projected, are capitalized. AllmiRNA sequences in Table I are derived from humans and are shown in 5′to 3′ orientation. Other miRNA sequences of the present invention can befound in the miRBase, the content of which is incorporated by referenceherein.

TABLE I miRBase SEQ ID miRNA name number Sequence NO hsa-let-7aMIMAT0000062 UGAGGUAGuagguuguauaguu 1 hsa-let-7a* MIMAT0004481CUAUACAAucuacugucuuuc 2 hsa-let-7a-2* MIMAT0010195CUGUACAGccuccuagcuuucc 3 hsa-let-7b MIMAT0000063 UGAGGUAGuagguugugugguu4 hsa-let-7b* MIMAT0004482 CUAUACAAccuacugccuuccc 5 hsa-let-7cMIMAT0000064 UGAGGUAGuagguuguaugguu 6 hsa-let-7c* MIMAT0004483UAGAGUUAcacccugggaguua 7 hsa-let-7d MIMAT0000065 AGAGGUAGuagguugcauaguu8 hsa-let-7d* MIMAT0004484 CUAUACGAccugcugccuuucu 9 hsa-let-7eMIMAT0000066 UGAGGUAGgagguuguauaguu 10 hsa-let-7e* MIMAT0004485CUAUACGGccuccuagcuuucc 11 hsa-let-7f MIMAT0000067 UGAGGUAGuagauuguauaguu12 hsa-let-7f-1* MIMAT0004486 CUAUACAAucuauugccuuccc 13 hsa-let-7f-2*MIMAT0004487 CUAUACAGucuacugucuuucc 14 hsa-miR-15a MIMAT0000068UAGCAGCAcauaaugguuugug 15 hsa-miR-15a* MIMAT0004488CAGGCCAUauugugcugccuca 16 hsa-miR-16 MIMAT0000069 UAGCAGCAcguaaauauuggcg17 hsa-miR-16-1* MIMAT0004489 CCAGUAUUaacugugcugcuga 18 hsa-miR-17MIMAT0000070 CAAAGUGCuuacagugcagguag 19 hsa-miR-17* MIMAT0000071ACUGCAGUgaaggcacuuguag 20 hsa-miR-18a MIMAT0000072UAAGGUGCaucuagugcagauag 21 hsa-miR-18a* MIMAT0002891ACUGCCCUaagugcuccuucugg 22 hsa-miR-19a* MIMAT0004490AGUUUUGCauaguugcacuaca 23 hsa-miR-19a MIMAT0000073UGUGCAAAucuaugcaaaacuga 24 hsa-miR-19b-1* MIMAT0004491AGUUUUGCagguuugcauccagc 25 hsa-miR-19b MIMAT0000074UGUGCAAAuccaugcaaaacuga 26 hsa-miR-19b-2* MIMAT0004492AGUUUUGCagguuugcauuuca 27 hsa-miR-20a MIMAT0000075UAAAGUGCuuauagugcagguag 28 hsa-miR-20a* MIMAT0004493ACUGCAUUaugagcacuuaaag 29 hsa-miR-21 MIMAT0000076 UAGCUUAUcagacugauguuga30 hsa-miR-21* MIMAT0004494 CAACACCAgucgaugggcugu 31 hsa-miR-22*MIMAT0004495 AGUUCUUCaguggcaagcuuua 32 hsa-miR-22 MIMAT0000077AAGCUGCCaguugaagaacugu 33 hsa-miR-23a* MIMAT0004496GGGGUUCCuggggaugggauuu 34 hsa-miR-23a MIMAT0000078 AUCACAUUgccagggauuucc35 hsa-miR-24-1* MIMAT0000079 UGCCUACUgagcugauaucagu 36 hsa-miR-24MIMAT0000080 UGGCUCAGuucagcaggaacag 37 hsa-miR-24-2* MIMAT0004497UGCCUACUgagcugaaacacag 38 hsa-miR-25* MIMAT0004498 AGGCGGAGacuugggcaauug39 hsa-miR-25 MIMAT0000081 CAUUGCACuugucucggucuga 40 hsa-miR-26aMIMAT0000082 UUCAAGUAauccaggauaggcu 41 hsa-miR-26a-1* MIMAT0004499CCUAUUCUugguuacuugcacg 42 hsa-miR-26b MIMAT0000083 UUCAAGUAauucaggauaggu43 hsa-miR-26b* MIMAT0004500 CCUGUUCUccauuacuuggcuc 44 hsa-miR-27a*MIMAT0004501 AGGGCUUAgcugcuugugagca 45 hsa-miR-27a MIMAT0000084UUCACAGUggcuaaguuccgc 46 hsa-miR-28-5p MIMAT0000085AAGGAGCUcacagucuauugag 47 hsa-miR-28-3p MIMAT0004502CACUAGAUugugagcuccugga 48 hsa-miR-29a* MIMAT0004503ACUGAUUUcuuuugguguucag 49 hsa-miR-29a MIMAT0000086UAGCACCAucugaaaucgguua 50 hsa-miR-30a MIMAT0000087UGUAAACAuccucgacuggaag 51 hsa-miR-30a* MIMAT0000088CUUUCAGUcggauguuugcagc 52 hsa-miR-31 MIMAT0000089 AGGCAAGAugcuggcauagcu53 hsa-miR-31* MIMAT0004504 UGCUAUGCcaacauauugccau 54 hsa-miR-32MIMAT0000090 UAUUGCACauuacuaaguugca 55 hsa-miR-32* MIMAT0004505CAAUUUAGugugugugauauuu 56 hsa-miR-33a MIMAT0000091 GUGCAUUGuaguugcauugca57 hsa-miR-33a* MIMAT0004506 CAAUGUUUccacagugcaucac 58 hsa-miR-92a-1*MIMAT0004507 AGGUUGGGaucgguugcaaugcu 59 hsa-miR-92a MIMAT0000092UAUUGCACuugucccggccugu 60 hsa-miR-92a-2* MIMAT0004508GGGUGGGGauuuguugcauuac 61 hsa-miR-93 MIMAT0000093CAAAGUGCuguucgugcagguag 62 hsa-miR-93* MIMAT0004509ACUGCUGAgcuagcacuucccg 63 hsa-miR-95 MIMAT0000094 UUCAACGGguauuuauugagca64 hsa-miR-96 MIMAT0000095 UUUGGCACuagcacauuuuugcu 65 hsa-miR-96*MIMAT0004510 AAUCAUGUgcagugccaauaug 66 hsa-miR-98 MIMAT0000096UGAGGUAGuaaguuguauuguu 67 hsa-miR-99a MIMAT0000097AACCCGUAgauccgaucuugug 68 hsa-miR-99a* MIMAT0004511CAAGCUCGcuucuaugggucug 69 hsa-miR-100 MIMAT0000098AACCCGUAgauccgaacuugug 70 hsa-miR-100* MIMAT0004512CAAGCUUGuaucuauagguaug 71 hsa-miR-101* MIMAT0004513CAGUUAUCacagugcugaugcu 72 hsa-miR-101 MIMAT0000099 UACAGUACugugauaacugaa73 hsa-miR-29b-1* MIMAT0004514 GCUGGUUUcauauggugguuuaga 74 hsa-miR-29bMIMAT0000100 UAGCACCAuuugaaaucaguguu 75 hsa-miR-29b-2* MIMAT0004515CUGGUUUCacaugguggcuuag 76 hsa-miR-103-2* MIMAT0009196AGCUUCUUuacagugcugccuug 77 hsa-miR-103 MIMAT0000101AGCAGCAUuguacagggcuauga 78 hsa-miR-105 MIMAT0000102UCAAAUGCucagacuccuguggu 79 hsa-miR-105* MIMAT0004516ACGGAUGUuugagcaugugcua 80 hsa-miR-106a MIMAT0000103AAAAGUGCuuacagugcagguag 81 hsa-miR-106a* MIMAT0004517CUGCAAUGuaagcacuucuuac 82 hsa-miR-107 MIMAT0000104AGCAGCAUuguacagggcuauca 83 hsa-miR-16-2* MIMAT0004518CCAAUAUUacugugcugcuuua 84 hsa-miR-192 MIMAT0000222 CUGACCUAugaauugacagcc85 hsa-miR-192* MIMAT0004543 CUGCCAAUuccauaggucacag 86 hsa-miR-196aMIMAT0000226 UAGGUAGUuucauguuguuggg 87 hsa-miR-197 MIMAT0000227UUCACCACcuucuccacccagc 88 hsa-miR-198 MIMAT0000228GGUCCAGAggggagauagguuc 89 hsa-miR-199a-5p MIMAT0000231CCCAGUGUucagacuaccuguuc 90 hsa-miR-199a-3p MIMAT0000232ACAGUAGUcugcacauugguua 91 hsa-miR-208a MIMAT0000241AUAAGACGagcaaaaagcuugu 92 hsa-miR-129-5p MIMAT0000242CUUUUUGCggucugggcuugc 93 hsa-miR-129* MIMAT0004548AAGCCCUUaccccaaaaaguau 94 hsa-miR-148a* MIMAT0004549AAAGUUCUgagacacuccgacu 95 hsa-miR-148a MIMAT0000243UCAGUGCAcuacagaacuuugu 96 hsa-miR-30c MIMAT0000244UGUAAACAuccuacacucucagc 97 hsa-miR-30c-2* MIMAT0004550CUGGGAGAaggcuguuuacucu 98 hsa-miR-30d MIMAT0000245UGUAAACAuccccgacuggaag 99 hsa-miR-30d* MIMAT0004551CUUUCAGUcagauguuugcugc 100 hsa-miR-139-5p MIMAT0000250UCUACAGUgcacgugucuccag 101 hsa-miR-139-3p MIMAT0004552GGAGACGCggcccuguuggagu 102 hsa-miR-147 MIMAT0000251 GUGUGUGGaaaugcuucugc103 hsa-miR-7 MIMAT0000252 UGGAAGACuagugauuuuguugu 104 hsa-miR-7-1*MIMAT0004553 CAACAAAUcacagucugccaua 105 hsa-miR-7-2* MIMAT0004554CAACAAAUcccagucuaccuaa 106 hsa-miR-10a MIMAT0000253UACCCUGUagauccgaauuugug 107 hsa-miR-10a* MIMAT0004555CAAAUUCGuaucuaggggaaua 108 hsa-miR-10b MIMAT0000254UACCCUGUagaaccgaauuugug 109 hsa-miR-10b* MIMAT0004556ACAGAUUCgauucuaggggaau 110 hsa-miR-34a MIMAT0000255UGGCAGUGucuuagcugguugu 111 hsa-miR-34a* MIMAT0004557CAAUCAGCaaguauacugcccu 112 hsa-miR-181a MIMAT0000256AACAUUCAacgcugucggugagu 113 hsa-miR-181a-2* MIMAT0004558ACCACUGAccguugacuguacc 114 hsa-miR-181b MIMAT0000257AACAUUCAuugcugucggugggu 115 hsa-miR-181c MIMAT0000258AACAUUCAaccugucggugagu 116 hsa-miR-181c* MIMAT0004559AACCAUCGaccguugaguggac 117 hsa-miR-182 MIMAT0000259UUUGGCAAugguagaacucacacu 118 hsa-miR-182* MIMAT0000260UGGUUCUAgacuugccaacua 119 hsa-miR-183 MIMAT0000261UAUGGCACugguagaauucacu 120 hsa-miR-183* MIMAT0004560GUGAAUUAccgaagggccauaa 121 hsa-miR-187* MIMAT0004561GGCUACAAcacaggacccgggc 122 hsa-miR-187 MIMAT0000262UCGUGUCUuguguugcagccgg 123 hsa-miR-196a* MIMAT0004562CGGCAACAagaaacugccugag 124 hsa-miR-199b-5p MIMAT0000263CCCAGUGUuuagacuaucuguuc 125 hsa-miR-199b-3p MIMAT0004563ACAGUAGUcugcacauugguua 91 hsa-miR-203 MIMAT0000264GUGAAAUGuuuaggaccacuag 126 hsa-miR-204 MIMAT0000265UUCCCUUUgucauccuaugccu 127 hsa-miR-205 MIMAT0000266UCCUUCAUuccaccggagucug 128 hsa-miR-205* MIMAT0009197GAUUUCAGuggagugaaguuc 129 hsa-miR-210 MIMAT0000267CUGUGCGUgugacagcggcuga 130 hsa-miR-211 MIMAT0000268UUCCCUUUgucauccuucgccu 131 hsa-miR-212 MIMAT0000269UAACAGUCuccagucacggcc 132 hsa-miR-181a* MIMAT0000270ACCAUCGAccguugauuguacc 133 hsa-miR-214* MIMAT0004564UGCCUGUCuacacuugcugugc 134 hsa-miR-214 MIMAT0000271ACAGCAGGcacagacaggcagu 135 hsa-miR-215 MIMAT0000272AUGACCUAugaauugacagac 136 hsa-miR-216a MIMAT0000273UAAUCUCAgcuggcaacuguga 137 hsa-miR-217 MIMAT0000274UACUGCAUcaggaacugauugga 138 hsa-miR-218 MIMAT0000275UUGUGCUUgaucuaaccaugu 139 hsa-miR-218-1* MIMAT0004565AUGGUUCCgucaagcaccaugg 140 hsa-miR-218-2* MIMAT0004566CAUGGUUCugucaagcaccgcg 141 hsa-miR-219-5p MIMAT0000276UGAUUGUCcaaacgcaauucu 142 hsa-miR-219-1-3p MIMAT0004567AGAGUUGAgucuggacgucccg 143 hsa-miR-220a MIMAT0000277CCACACCGuaucugacacuuu 144 hsa-miR-221* MIMAT0004568ACCUGGCAuacaauguagauuu 145 hsa-miR-221 MIMAT0000278AGCUACAUugucugcuggguuuc 146 hsa-miR-222* MIMAT0004569CUCAGUAGccaguguagauccu 147 hsa-miR-222 MIMAT0000279AGCUACAUcuggcuacugggu 148 hsa-miR-223* MIMAT0004570CGUGUAUUugacaagcugaguu 149 hsa-miR-223 MIMAT0000280UGUCAGUUugucaaauacccca 150 hsa-miR-224 MIMAT0000281CAAGUCACuagugguuccguu 151 hsa-miR-224* MIMAT0009198AAAAUGGUgcccuagugacuaca 152 hsa-miR-200b* MIMAT0004571CAUCUUACugggcagcauugga 153 hsa-miR-200b MIMAT0000318UAAUACUGccugguaaugauga 154 hsa-let-7g MIMAT0000414UGAGGUAGuaguuuguacaguu 155 hsa-let-7g* MIMAT0004584CUGUACAGgccacugccuugc 156 hsa-let-7i MIMAT0000415 UGAGGUAGuaguuugugcuguu157 hsa-let-7i* MIMAT0004585 CUGCGCAAgcuacugccuugcu 158 hsa-miR-1MIMAT0000416 UGGAAUGUaaagaaguauguau 159 hsa-miR-15b MIMAT0000417UAGCAGCAcaucaugguuuaca 160 hsa-miR-15b* MIMAT0004586CGAAUCAUuauuugcugcucua 161 hsa-miR-23b* MIMAT0004587UGGGUUCCuggcaugcugauuu 162 hsa-miR-23b MIMAT0000418AUCACAUUgccagggauuacc 163 hsa-miR-27b* MIMAT0004588AGAGCUUAgcugauuggugaac 164 hsa-miR-27b MIMAT0000419UUCACAGUggcuaaguucugc 165 hsa-miR-30b MIMAT0000420UGUAAACAuccuacacucagcu 166 hsa-miR-30b* MIMAT0004589CUGGGAGGuggauguuuacuuc 167 hsa-miR-122 MIMAT0000421UGGAGUGUgacaaugguguuug 168 hsa-miR-122* MIMAT0004590AACGCCAUuaucacacuaaaua 169 hsa-miR-124* MIMAT0004591CGUGUUCAcagcggaccuugau 170 hsa-miR-124 MIMAT0000422 UAAGGCACgcggugaaugcc171 hsa-miR-125b MIMAT0000423 UCCCUGAGacccuaacuuguga 172 hsa-miR-125b-1*MIMAT0004592 ACGGGUUAggcucuugggagcu 173 hsa-miR-128 MIMAT0000424UCACAGUGaaccggucucuuu 174 hsa-miR-130a* MIMAT0004593UUCACAUUgugcuacugucugc 175 hsa-miR-130a MIMAT0000425CAGUGCAAuguuaaaagggcau 176 hsa-miR-132* MIMAT0004594ACCGUGGCuuucgauuguuacu 177 hsa-miR-132 MIMAT0000426UAACAGUCuacagccauggucg 178 hsa-miR-133a MIMAT0000427UUUGGUCCccuucaaccagcug 179 hsa-miR-135a MIMAT0000428UAUGGCUUuuuauuccuauguga 180 hsa-miR-135a* MIMAT0004595UAUAGGGAuuggagccguggcg 181 hsa-miR-137 MIMAT0000429UUAUUGCUuaagaauacgcguag 182 hsa-miR-138 MIMAT0000430AGCUGGUGuugugaaucaggccg 183 hsa-miR-138-2* MIMAT0004596GCUAUUUCacgacaccaggguu 184 hsa-miR-140-5p MIMAT0000431CAGUGGUUuuacccuaugguag 185 hsa-miR-140-3p MIMAT0004597UACCACAGgguagaaccacgg 186 hsa-miR-141* MIMAT0004598CAUCUUCCaguacaguguugga 187 hsa-miR-141 MIMAT0000432UAACACUGucugguaaagaugg 188 hsa-miR-142-5p MIMAT0000433CAUAAAGUagaaagcacuacu 189 hsa-miR-142-3p MIMAT0000434UGUAGUGUuuccuacuuuaugga 190 hsa-miR-143* MIMAT0004599GGUGCAGUgcugcaucucuggu 191 hsa-miR-143 MIMAT0000435UGAGAUGAagcacuguagcuc 192 hsa-miR-144* MIMAT0004600GGAUAUCAucauauacuguaag 193 hsa-miR-144 MIMAT0000436 UACAGUAUagaugauguacu194 hsa-miR-145 MIMAT0000437 GUCCAGUUuucccaggaaucccu 195 hsa-miR-145*MIMAT0004601 GGAUUCCUggaaauacuguucu 196 hsa-miR-152 MIMAT0000438UCAGUGCAugacagaacuugg 197 hsa-miR-153 MIMAT0000439UUGCAUAGucacaaaagugauc 198 hsa-miR-191 MIMAT0000440CAACGGAAucccaaaagcagcug 199 hsa-miR-191* MIMAT0001618GCUGCGCUuggauuucgucccc 200 hsa-miR-9 MIMAT0000441UCUUUGGUuaucuagcuguauga 201 hsa-miR-9* MIMAT0000442AUAAAGCUagauaaccgaaagu 202 hsa-miR-125a-5p MIMAT0000443UCCCUGAGacccuuuaaccuguga 203 hsa-miR-125a-3p MIMAT0004602ACAGGUGAgguucuugggagcc 204 hsa-miR-125b-2* MIMAT0004603UCACAAGUcaggcucuugggac 205 hsa-miR-126* MIMAT0000444CAUUAUUAcuuuugguacgcg 206 hsa-miR-126 MIMAT0000445UCGUACCGugaguaauaaugcg 207 hsa-miR-127-5p MIMAT0004604CUGAAGCUcagagggcucugau 208 hsa-miR-127-3p MIMAT0000446UCGGAUCCgucugagcuuggcu 209 hsa-miR-129-3p MIMAT0004605AAGCCCUUaccccaaaaagcau 210 hsa-miR-134 MIMAT0000447UGUGACUGguugaccagagggg 211 hsa-miR-136 MIMAT0000448ACUCCAUUuguuuugaugaugga 212 hsa-miR-136* MIMAT0004606CAUCAUCGucucaaaugagucu 213 hsa-miR-138-1* MIMAT0004607GCUACUUCacaacaccagggcc 214 hsa-miR-146a MIMAT0000449UGAGAACUgaauuccauggguu 215 hsa-miR-146a* MIMAT0004608CCUCUGAAauucaguucuucag 216 hsa-miR-149 MIMAT0000450UCUGGCUCcgugucuucacuccc 217 hsa-miR-149* MIMAT0004609AGGGAGGGacgggggcugugc 218 hsa-miR-150 MIMAT0000451UCUCCCAAcccuuguaccagug 219 hsa-miR-150* MIMAT0004610CUGGUACAggccugggggacag 220 hsa-miR-154 MIMAT0000452UAGGUUAUccguguugccuucg 221 hsa-miR-154* MIMAT0000453AAUCAUACacgguugaccuauu 222 hsa-miR-184 MIMAT0000454UGGACGGAgaacugauaagggu 223 hsa-miR-185 MIMAT0000455UGGAGAGAaaggcaguuccuga 224 hsa-miR-185* MIMAT0004611AGGGGCUGgcuuuccucugguc 225 hsa-miR-186 MIMAT0000456CAAAGAAUucuccuuuugggcu 226 hsa-miR-186* MIMAT0004612GCCCAAAGgugaauuuuuuggg 227 hsa-miR-188-5p MIMAT0000457CAUCCCUUgcaugguggaggg 228 hsa-miR-188-3p MIMAT0004613CUCCCACAugcaggguuugca 229 hsa-miR-190 MIMAT0000458UGAUAUGUuugauauauuaggu 230 hsa-miR-193a-5p MIMAT0004614UGGGUCUUugcgggcgagauga 231 hsa-miR-193a-3p MIMAT0000459AACUGGCCuacaaagucccagu 232 hsa-miR-194 MIMAT0000460UGUAACAGcaacuccaugugga 233 hsa-miR-195 MIMAT0000461UAGCAGCAcagaaauauuggc 234 hsa-miR-195* MIMAT0004615CCAAUAUUggcugugcugcucc 235 hsa-miR-206 MIMAT0000462UGGAAUGUaaggaagugugugg 236 hsa-miR-320a MIMAT0000510AAAAGCUGgguugagagggcga 237 hsa-miR-200c* MIMAT0004657CGUCUUACccagcaguguuugg 238 hsa-miR-200c MIMAT0000617UAAUACUGccggguaaugaugga 239 hsa-miR-155 MIMAT0000646UUAAUGCUaaucgugauaggggu 240 hsa-miR-155* MIMAT0004658CUCCUACAuauuagcauuaaca 241 hsa-miR-194* MIMAT0004671CCAGUGGGgcugcuguuaucug 242 hsa-miR-106b MIMAT0000680UAAAGUGCugacagugcagau 243 hsa-miR-106b* MIMAT0004672CCGCACUGuggguacuugcugc 244 hsa-miR-29c* MIMAT0004673UGACCGAUuucuccugguguuc 245 hsa-miR-29c MIMAT0000681UAGCACCAuuugaaaucgguua 246 hsa-miR-30c-1* MIMAT0004674CUGGGAGAggguuguuuacucc 247 hsa-miR-200a* MIMAT0001620CAUCUUACcggacagugcugga 248 hsa-miR-200a MIMAT0000682UAACACUGucugguaacgaugu 249 hsa-miR-302a* MIMAT0000683ACUUAAACguggauguacuugcu 250 hsa-miR-302a MIMAT0000684UAAGUGCUuccauguuuugguga 251 hsa-miR-219-2-3p MIMAT0004675AGAAUUGUggcuggacaucugu 252 hsa-miR-34b* MIMAT0000685UAGGCAGUgucauuagcugauug 253 hsa-miR-34b MIMAT0004676CAAUCACUaacuccacugccau 254 hsa-miR-34c-5p MIMAT0000686AGGCAGUGuaguuagcugauugc 255 hsa-miR-34c-3p MIMAT0004677AAUCACUAaccacacggccagg 256 hsa-miR-299-5p MIMAT0002890UGGUUUACcgucccacauacau 257 hsa-miR-299-3p MIMAT0000687UAUGUGGGaugguaaaccgcuu 258 hsa-miR-301a MIMAT0000688CAGUGCAAuaguauugucaaagc 259 hsa-miR-99b MIMAT0000689CACCCGUAgaaccgaccuugcg 260 hsa-miR-99b* MIMAT0004678CAAGCUCGugucuguggguccg 261 hsa-miR-296-5p MIMAT0000690AGGGCCCCcccucaauccugu 262 hsa-miR-296-3p MIMAT0004679GAGGGUUGgguggaggcucucc 263 hsa-miR-130b* MIMAT0004680ACUCUUUCccuguugcacuac 264 hsa-miR-130b MIMAT0000691CAGUGCAAugaugaaagggcau 265 hsa-miR-30e MIMAT0000692UGUAAACAuccuugacuggaag 266 hsa-miR-30e* MIMAT0000693CUUUCAGUcggauguuuacagc 267 hsa-miR-26a-2* MIMAT0004681CCUAUUCUugauuacuuguuuc 268 hsa-miR-361-5p MIMAT0000703UUAUCAGAaucuccagggguac 269 hsa-miR-361-3p MIMAT0004682UCCCCCAGgugugauucugauuu 270 hsa-miR-362-5p MIMAT0000705AAUCCUUGgaaccuaggugugagu 271 hsa-miR-362-3p MIMAT0004683AACACACCuauucaaggauuca 272 hsa-miR-363* MIMAT0003385CGGGUGGAucacgaugcaauuu 273 hsa-miR-363 MIMAT0000707AAUUGCACgguauccaucugua 274 hsa-miR-365 MIMAT0000710UAAUGCCCcuaaaaauccuuau 275 hsa-miR-365* MIMAT0009199AGGGACUUucaggggcagcugu 276 hsa-miR-302b* MIMAT0000714ACUUUAACauggaagugcuuuc 277 hsa-miR-302b MIMAT0000715UAAGUGCUuccauguuuuaguag 278 hsa-miR-302c* MIMAT0000716UUUAACAUggggguaccugcug 279 hsa-miR-302c MIMAT0000717UAAGUGCUuccauguuucagugg 280 hsa-miR-302d* MIMAT0004685ACUUUAACauggaggcacuugc 281 hsa-miR-302d MIMAT0000718UAAGUGCUuccauguuugagugu 282 hsa-miR-367* MIMAT0004686ACUGUUGCuaauaugcaacucu 283 hsa-miR-367 MIMAT0000719AAUUGCACuuuagcaaugguga 284 hsa-miR-376c MIMAT0000720AACAUAGAggaaauuccacgu 285 hsa-miR-369-5p MIMAT0001621AGAUCGACcguguuauauucgc 286 hsa-miR-369-3p MIMAT0000721AAUAAUACaugguugaucuuu 287 hsa-miR-370 MIMAT0000722GCCUGCUGggguggaaccuggu 288 hsa-miR-371-5p MIMAT0004687ACUCAAACugugggggcacu 289 hsa-miR-371-3p MIMAT0000723AAGUGCCGccaucuuuugagugu 290 hsa-miR-372 MIMAT0000724AAAGUGCUgcgacauuugagcgu 291 hsa-miR-373* MIMAT0000725ACUCAAAAugggggcgcuuucc 292 hsa-miR-373 MIMAT0000726GAAGUGCUucgauuuuggggugu 293 hsa-miR-374a MIMAT0000727UUAUAAUAcaaccugauaagug 294 hsa-miR-374a* MIMAT0004688CUUAUCAGauuguauuguaauu 295 hsa-miR-375 MIMAT0000728UUUGUUCGuucggcucgcguga 296 hsa-miR-376a* MIMAT0003386GUAGAUUCuccuucuaugagua 297 hsa-miR-376a MIMAT0000729AUCAUAGAggaaaauccacgu 298 hsa-miR-377* MIMAT0004689AGAGGUUGcccuuggugaauuc 299 hsa-miR-377 MIMAT0000730AUCACACAaaggcaacuuuugu 300 hsa-miR-378* MIMAT0000731CUCCUGACuccagguccugugu 301 hsa-miR-378 MIMAT0000732ACUGGACUuggagucagaagg 302 hsa-miR-379 MIMAT0000733 UGGUAGACuauggaacguagg303 hsa-miR-379* MIMAT0004690 UAUGUAACaugguccacuaacu 304 hsa-miR-380*MIMAT0000734 UGGUUGACcauagaacaugcgc 305 hsa-miR-380 MIMAT0000735UAUGUAAUaugguccacaucuu 306 hsa-miR-381 MIMAT0000736UAUACAAGggcaagcucucugu 307 hsa-miR-382 MIMAT0000737GAAGUUGUucgugguggauucg 308 hsa-miR-383 MIMAT0000738AGAUCAGAaggugauuguggcu 309 hsa-miR-340 MIMAT0004692UUAUAAAGcaaugagacugauu 310 hsa-miR-340* MIMAT0000750UCCGUCUCaguuacuuuauagc 311 hsa-miR-330-5p MIMAT0004693UCUCUGGGccugugucuuaggc 312 hsa-miR-330-3p MIMAT0000751GCAAAGCAcacggccugcagaga 313 hsa-miR-328 MIMAT0000752CUGGCCCUcucugcccuuccgu 314 hsa-miR-342-5p MIMAT0004694AGGGGUGCuaucugugauuga 315 hsa-miR-342-3p MIMAT0000753UCUCACACagaaaucgcacccgu 316 hsa-miR-337-5p MIMAT0004695GAACGGCUucauacaggaguu 317 hsa-miR-337-3p MIMAT0000754CUCCUAUAugaugccuuucuuc 318 hsa-miR-323-5p MIMAT0004696AGGUGGUCcguggcgcguucgc 319 hsa-miR-323-3p MIMAT0000755CACAUUACacggucgaccucu 320 hsa-miR-326 MIMAT0000756 CCUCUGGGcccuuccuccag321 hsa-miR-151-5p MIMAT0004697 UCGAGGAGcucacagucuagu 322 hsa-miR-151-3pMIMAT0000757 CUAGACUGaagcuccuugagg 323 hsa-miR-135b MIMAT0000758UAUGGCUUuucauuccuauguga 324 hsa-miR-135b* MIMAT0004698AUGUAGGGcuaaaagccauggg 325 hsa-miR-148b* MIMAT0004699AAGUUCUGuuauacacucaggc 326 hsa-miR-148b MIMAT0000759UCAGUGCAucacagaacuuugu 327 hsa-miR-331-5p MIMAT0004700CUAGGUAUggucccagggaucc 328 hsa-miR-331-3p MIMAT0000760GCCCCUGGgccuauccuagaa 329 hsa-miR-324-5p MIMAT0000761CGCAUCCCcuagggcauuggugu 330 hsa-miR-324-3p MIMAT0000762ACUGCCCCaggugcugcugg 331 hsa-miR-338-5p MIMAT0004701AACAAUAUccuggugcugagug 332 hsa-miR-338-3p MIMAT0000763UCCAGCAUcagugauuuuguug 333 hsa-miR-339-5p MIMAT0000764UCCCUGUCcuccaggagcucacg 334 hsa-miR-339-3p MIMAT0004702UGAGCGCCucgacgacagagccg 335 hsa-miR-335 MIMAT0000765UCAAGAGCaauaacgaaaaaugu 336 hsa-miR-335* MIMAT0004703UUUUUCAUuauugcuccugacc 337 hsa-miR-133b MIMAT0000770UUUGGUCCccuucaaccagcua 338 hsa-miR-325 MIMAT0000771CCUAGUAGguguccaguaagugu 339 hsa-miR-345 MIMAT0000772GCUGACUCcuaguccagggcuc 340 hsa-miR-346 MIMAT0000773UGUCUGCCcgcaugccugccucu 341 hsa-miR-384 MIMAT0001075AUUCCUAGaaauuguucaua 342 hsa-miR-196b MIMAT0001080UAGGUAGUuuccuguuguuggg 343 hsa-miR-196b* MIMAT0009201UCGACAGCacgacacugccuuc 344 hsa-miR-422a MIMAT0001339ACUGGACUuagggucagaaggc 345 hsa-miR-423-5p MIMAT0004748UGAGGGGCagagagcgagacuuu 346 hsa-miR-423-3p MIMAT0001340AGCUCGGUcugaggccccucagu 347 hsa-miR-424 MIMAT0001341CAGCAGCAauucauguuuugaa 348 hsa-miR-424* MIMAT0004749CAAAACGUgaggcgcugcuau 349 hsa-miR-425 MIMAT0003393AAUGACACgaucacucccguuga 350 hsa-miR-425* MIMAT0001343AUCGGGAAugucguguccgccc 351 hsa-miR-18b MIMAT0001412UAAGGUGCaucuagugcaguuag 352 hsa-miR-18b* MIMAT0004751UGCCCUAAaugccccuucuggc 353 hsa-miR-20b MIMAT0001413CAAAGUGCucauagugcagguag 354 hsa-miR-20b* MIMAT0004752ACUGUAGUaugggcacuuccag 355 hsa-miR-448 MIMAT0001532UUGCAUAUguaggaugucccau 356 hsa-miR-429 MIMAT0001536UAAUACUGucugguaaaaccgu 357 hsa-miR-449a MIMAT0001541UGGCAGUGuauuguuagcuggu 358 hsa-miR-450a MIMAT0001545UUUUGCGAuguguuccuaauau 359 hsa-miR-431 MIMAT0001625UGUCUUGCaggccgucaugca 360 hsa-miR-431* MIMAT0004757CAGGUCGUcuugcagggcuucu 361 hsa-miR-433 MIMAT0001627AUCAUGAUgggcuccucggugu 362 hsa-miR-329 MIMAT0001629AACACACCugguuaaccucuuu 363 hsa-miR-451 MIMAT0001631AAACCGUUaccauuacugaguu 364 hsa-miR-452 MIMAT0001635AACUGUUUgcagaggaaacuga 365 hsa-miR-452* MIMAT0001636CUCAUCUGcaaagaaguaagug 366 hsa-miR-409-5p MIMAT0001638AGGUUACCcgagcaacuuugcau 367 hsa-miR-409-3p MIMAT0001639GAAUGUUGcucggugaaccccu 368 hsa-miR-412 MIMAT0002170ACUUCACCugguccacuagccgu 369 hsa-miR-410 MIMAT0002171AAUAUAACacagauggccugu 370 hsa-miR-376b MIMAT0002172AUCAUAGAggaaaauccauguu 371 hsa-miR-483-5p MIMAT0004761AAGACGGGaggaaagaagggag 372 hsa-miR-483-3p MIMAT0002173UCACUCCUcuccucccgucuu 373 hsa-miR-484 MIMAT0002174UCAGGCUCaguccccucccgau 374 hsa-miR-485-5p MIMAT0002175AGAGGCUGgccgugaugaauuc 375 hsa-miR-485-3p MIMAT0002176GUCAUACAcggcucuccucucu 376 hsa-miR-486-5p MIMAT0002177UCCUGUACugagcugccccgag 377 hsa-miR-486-3p MIMAT0004762CGGGGCAGcucaguacaggau 378 hsa-miR-487a MIMAT0002178AAUCAUACagggacauccaguu 379 hsa-miR-488* MIMAT0002804CCCAGAUAauggcacucucaa 380 hsa-miR-488 MIMAT0004763 UUGAAAGGcuauuucuugguc381 hsa-miR-489 MIMAT0002805 GUGACAUCacauauacggcagc 382 hsa-miR-490-5pMIMAT0004764 CCAUGGAUcuccaggugggu 383 hsa-miR-490-3p MIMAT0002806CAACCUGGaggacuccaugcug 384 hsa-miR-491-5p MIMAT0002807AGUGGGGAacccuuccaugagg 385 hsa-miR-491-3p MIMAT0004765CUUAUGCAagauucccuucuac 386 hsa-miR-511 MIMAT0002808GUGUCUUUugcucugcaguca 387 hsa-miR-146b-5p MIMAT0002809UGAGAACUgaauuccauaggcu 388 hsa-miR-146b-3p MIMAT0004766UGCCCUGUggacucaguucugg 389 hsa-miR-202* MIMAT0002810UUCCUAUGcauauacuucuuug 390 hsa-miR-202 MIMAT0002811 AGAGGUAUagggcaugggaa391 hsa-miR-492 MIMAT0002812 AGGACCUGcgggacaagauucuu 392 hsa-miR-493*MIMAT0002813 UUGUACAUgguaggcuuucauu 393 hsa-miR-493 MIMAT0003161UGAAGGUCuacugugugccagg 394 hsa-miR-432 MIMAT0002814UCUUGGAGuaggucauugggugg 395 hsa-miR-432* MIMAT0002815CUGGAUGGcuccuccaugucu 396 hsa-miR-494 MIMAT0002816UGAAACAUacacgggaaaccuc 397 hsa-miR-495 MIMAT0002817AAACAAACauggugcacuucuu 398 hsa-miR-496 MIMAT0002818UGAGUAUUacauggccaaucuc 399 hsa-miR-193b* MIMAT0004767CGGGGUUUugagggcgagauga 400 hsa-miR-193b MIMAT0002819AACUGGCCcucaaagucccgcu 401 hsa-miR-497 MIMAT0002820CAGCAGCAcacugugguuugu 402 hsa-miR-497* MIMAT0004768CAAACCACacugugguguuaga 403 hsa-miR-181d MIMAT0002821AACAUUCAuuguugucggugggu 404 hsa-miR-512-5p MIMAT0002822CACUCAGCcuugagggcacuuuc 405 hsa-miR-512-3p MIMAT0002823AAGUGCUGucauagcugagguc 406 hsa-miR-498 MIMAT0002824UUUCAAGCcagggggccfuuuuuc 407 hsa-miR-520e MIMAT0002825AAAGUGCUuccuuuuugaggg 408 hsa-miR-515-5p MIMAT0002826UUCUCCAAaagaaagcacuuucug 409 hsa-miR-515-3p MIMAT0002827GAGUGCCUucuuuuggagcguu 410 hsa-miR-519e* MIMAT0002828UUCUCCAAaagggagcacuuuc 411 hsa-miR-519e MIMAT0002829AAGUGCCUccuuuuagaguguu 412 hsa-miR-520f MIMAT0002830AAGUGCUUccuuuuagaggguu 413 hsa-miR-519c-5p MIMAT0002831CUCUAGAGggaagcgcuuucug 414 hsa-miR-519c-3p MIMAT0002832AAAGUGCAucuuuuuagaggau 415 hsa-miR-520a-5p MIMAT0002833CUCCAGAGggaaguacuuucu 416 hsa-miR-520a-3p MIMAT0002834AAAGUGCUucccuuuggacugu 417 hsa-miR-526b MIMAT0002835CUCUUGAGggaagcacuuucugu 418 hsa-miR-526b* MIMAT0002836GAAAGUGCuuccuuuuagaggc 419 hsa-miR-519b-5p MIMAT0005454CUCUAGAGggaagcgcuuucug 414 hsa-miR-519b-3p MIMAT0002837AAAGUGCAuccuuuuagagguu 420 hsa-miR-525-5p MIMAT0002838CUCCAGAGggaugcacuuucu 421 hsa-miR-525-3p MIMAT0002839GAAGGCGCuucccuuuagagcg 422 hsa-miR-523* MIMAT0005449CUCUAGAGggaagcgcuuucug 414 hsa-miR-523 MIMAT0002840GAACGCGCuucccuauagagggu 423 hsa-miR-518f* MIMAT0002841CUCUAGAGggaagcacuuucuc 424 hsa-miR-518f MIMAT0002842GAAAGCGCuucucuuuagagg 425 hsa-miR-520b MIMAT0002843AAAGUGCUuccuuuuagaggg 426 hsa-miR-518b MIMAT0002844CAAAGCGCuccccuuuagaggu 427 hsa-miR-526a MIMAT0002845CUCUAGAGggaagcacuuucug 428 hsa-miR-520c-5p MIMAT0005455CUCUAGAGggaagcacuuucug 428 hsa-miR-520c-3p MIMAT0002846AAAGUGCUuccuuuuagagggu 429 hsa-miR-518c* MIMAT0002847UCUCUGGAgggaagcacuuucug 430 hsa-miR-518c MIMAT0002848CAAAGCGCuucucuuuagagugu 431 hsa-miR-524-5p MIMAT0002849CUACAAAGggaagcacuuucuc 432 hsa-miR-524-3p MIMAT0002850GAAGGCGCuucccuuuggagu 433 hsa-miR-517* MIMAT0002851CCUCUAGAuggaagcacugucu 434 hsa-miR-517a MIMAT0002852AUCGUGCAucccuuuagagugu 435 hsa-miR-519d MIMAT0002853CAAAGUGCcucccuuuagagug 436 hsa-miR-521 MIMAT0002854AACGCACUucccuuuagagugu 437 hsa-miR-520d-5p MIMAT0002855CUACAAAGggaagcccuuuc 438 hsa-miR-520d-3p MIMAT0002856AAAGUGCUucucuuuggugggu 439 hsa-miR-517b MIMAT0002857UCGUGCAUcccuuuagaguguu 440 hsa-miR-520g MIMAT0002858ACAAAGUGcuucccuuuagagugu 441 hsa-miR-516b MIMAT0002859AUCUGGAGguaagaagcacuuu 442 hsa-miR-516b* MIMAT0002860 UGCUUCCUuucagagggu443 hsa-miR-518e* MIMAT0005450 CUCUAGAGggaagcgcuuucug 414 hsa-miR-518eMIMAT0002861 AAAGCGCUucccuucagagug 444 hsa-miR-518a-5p MIMAT0005457CUGCAAAGggaagcccuuuc 445 hsa-miR-518a-3p MIMAT0002863GAAAGCGCuucccuuugcugga 446 hsa-miR-518d-5p MIMAT0005456CUCUAGAGggaagcacuuucug 428 hsa-miR-518d-3p MIMAT0002864CAAAGCGCuucccuuuggagc 447 hsa-miR-517c MIMAT0002866AUCGUGCAuccuuuuagagugu 448 hsa-miR-520h MIMAT0002867ACAAAGUGcuucccuuuagagu 449 hsa-miR-522* MIMAT0005451CUCUAGAGggaagcgcuuucug 414 hsa-miR-522 MIMAT0002868AAAAUGGUucccuuuagagugu 450 hsa-miR-519a* MIMAT0005452CUCUAGAGggaagcgcuuucug 414 hsa-miR-519a MIMAT0002869AAAGUGCAuccuuuuagagugu 451 hsa-miR-527 MIMAT0002862 CUGCAAAGggaagcccuuuc445 hsa-miR-516a-5p MIMAT0004770 UUCUCGAGgaaagaagcacuuuc 452hsa-miR-516a-3p MIMAT0006778 UGCUUCCUuucagagggu 443 hsa-miR-499-5pMIMAT0002870 UUAAGACUugcagugauguuu 453 hsa-miR-499-3p MIMAT0004772AACAUCACagcaagucugugcu 454 hsa-miR-500 MIMAT0004773UAAUCCUUgcuaccugggugaga 455 hsa-miR-500* MIMAT0002871AUGCACCUgggcaaggauucug 456 hsa-miR-501-5p MIMAT0002872AAUCCUUUgucccugggugaga 457 hsa-miR-501-3p MIMAT0004774AAUGCACCcgggcaaggauucu 458 hsa-miR-502-5p MIMAT0002873AUCCUUGCuaucugggugcua 459 hsa-miR-502-3p MIMAT0004775AAUGCACCugggcaaggauuca 460 hsa-miR-503 MIMAT0002874UAGCAGCGggaacaguucugcag 461 hsa-miR-504 MIMAT0002875AGACCCUGgucugcacucuauc 462 hsa-miR-505* MIMAT0004776GGGAGCCAggaaguauugaugu 463 hsa-miR-505 MIMAT0002876CGUCAACAcuugcugguuuccu 464 hsa-miR-513a-5p MIMAT0002877UUCACAGGgaggugucau 465 hsa-miR-513a-3p MIMAT0004777UAAAUUUCaccuuucugagaagg 466 hsa-miR-506 MIMAT0002878UAAGGCACccuucugaguaga 467 hsa-miR-507 MIMAT0002879 UUUUGCACcuuuuggagugaa468 hsa-miR-508-5p MIMAT0004778 UACUCCAGagggcgucacucaug 469hsa-miR-508-3p MIMAT0002880 UGAUUGUAgccuuuuggaguaga 470 hsa-miR-509-5pMIMAT0004779 UACUGCAGacaguggcaauca 471 hsa-miR-509-3p MIMAT0002881UGAUUGGUacgucuguggguag 472 hsa-miR-510 MIMAT0002882UACUCAGGagaguggcaaucac 473 hsa-miR-514 MIMAT0002883AUUGACACuucugugaguaga 474 hsa-miR-532-5p MIMAT0002888CAUGCCUUgaguguaggaccgu 475 hsa-miR-532-3p MIMAT0004780CCUCCCACacccaaggcuugca 476 hsa-miR-455-5p MIMAT0003150UAUGUGCCuuuggacuacaucg 477 hsa-miR-455-3p MIMAT0004784GCAGUCCAugggcauauacac 478 hsa-miR-539 MIMAT0003163GGAGAAAUuauccuuggugugu 479 hsa-miR-544 MIMAT0003164AUUCUGCAuuuuuagcaaguuc 480 hsa-miR-545* MIMAT0004785UCAGUAAAuguuuauuagauga 481 hsa-miR-545 MIMAT0003165UCAGCAAAcauuuauugugugc 482 hsa-miR-487b MIMAT0003180AAUCGUACagggucauccacuu 483 hsa-miR-551a MIMAT0003214GCGACCCAcucuugguuucca 484 hsa-miR-552 MIMAT0003215 AACAGGUGacugguuagacaa485 hsa-miR-553 MIMAT0003216 AAAACGGUgagauuuuguuuu 486 hsa-miR-554MIMAT0003217 GCUAGUCCugacucagccagu 487 hsa-miR-92b* MIMAT0004792AGGGACGGgacgcggugcagug 488 hsa-miR-92b MIMAT0003218UAUUGCACucgucccggccucc 489 hsa-miR-555 MIMAT0003219AGGGUAAGcugaaccucugau 490 hsa-miR-556-5p MIMAT0003220GAUGAGCUcauuguaauaugag 491 hsa-miR-556-3p MIMAT0004793AUAUUACCauuagcucaucuuu 492 hsa-miR-557 MIMAT0003221GUUUGCACgggugggccuugucu 493 hsa-miR-558 MIMAT0003222 UGAGCUGCuguaccaaaau494 hsa-miR-559 MIMAT0003223 UAAAGUAAauaugcaccaaaa 495 hsa-miR-561MIMAT0003225 CAAAGUUUaagauccuugaagu 496 hsa-miR-562 MIMAT0003226AAAGUAGCuguaccauuugc 497 hsa-miR-563 MIMAT0003227 AGGUUGACauacguuuccc498 hsa-miR-564 MIMAT0003228 AGGCACGGugucagcaggc 499 hsa-miR-566MIMAT0003230 GGGCGCCUgugaucccaac 500 hsa-miR-567 MIMAT0003231AGUAUGUUcuuccaggacagaac 501 hsa-miR-568 MIMAT0003232AUGUAUAAauguauacacac 502 hsa-miR-551b* MIMAT0004794GAAAUCAAgcgugggugagacc 503 hsa-miR-551b MIMAT0003233GCGACCCAuacuugguuucag 504 hsa-miR-569 MIMAT0003234 AGUUAAUGaauccuggaaagu505 hsa-miR-570 MIMAT0003235 CGAAAACAgcaauuaccuuugc 506 hsa-miR-571MIMAT0003236 UGAGUUGGccaucugagugag 507 hsa-miR-572 MIMAT0003237GUCCGCUCggcgguggccca 508 hsa-miR-573 MIMAT0003238CUGAAGUGauguguaacugaucag 509 hsa-miR-574-5p MIMAT0004795UGAGUGUGugugugugagugugu 510 hsa-miR-574-3p MIMAT0003239CACGCUCAugcacacacccaca 511 hsa-miR-575 MIMAT0003240 GAGCCAGUuggacaggagc512 hsa-miR-576-5p MIMAT0003241 AUUCUAAUuucuccacgucuuu 513hsa-miR-576-3p MIMAT0004796 AAGAUGUGgaaaaauuggaauc 514 hsa-miR-577MIMAT0003242 UAGAUAAAauauugguaccug 515 hsa-miR-578 MIMAT0003243CUUCUUGUgcucuaggauugu 516 hsa-miR-579 MIMAT0003244UUCAUUUGguauaaaccgcgauu 517 hsa-miR-580 MIMAT0003245UUGAGAAUgaugaaucauuagg 518 hsa-miR-581 MIMAT0003246UCUUGUGUucucuagaucagu 519 hsa-miR-582-5p MIMAT0003247UUACAGUUguucaaccaguuacu 520 hsa-miR-582-3p MIMAT0004797UAACUGGUugaacaacugaacc 521 hsa-miR-583 MIMAT0003248CAAAGAGGaaggucccauuac 522 hsa-miR-584 MIMAT0003249UUAUGGUUugccugggacugag 523 hsa-miR-585 MIMAT0003250 UGGGCGUAucuguaugcua524 hsa-miR-548a-3p MIMAT0003251 CAAAACUGgcaauuacuuuugc 525 hsa-miR-586MIMAT0003252 UAUGCAUUguauuuuuaggucc 526 hsa-miR-587 MIMAT0003253UUUCCAUAggugaugagucac 527 hsa-miR-548b-5p MIMAT0004798AAAAGUAAuugugguuuuggcc 528 hsa-miR-548b-3p MIMAT0003254CAAGAACCucaguugcuuuugu 529 hsa-miR-588 MIMAT0003255UUGGCCACaauggguuagaac 530 hsa-miR-589 MIMAT0004799UGAGAACCacgucugcucugag 531 hsa-miR-589* MIMAT0003256UCAGAACAaaugccgguucccaga 532 hsa-miR-550 MIMAT0004800AGUGCCUGagggaguaagagccc 533 hsa-miR-550* MIMAT0003257UGUCUUACucccucaggcacau 534 hsa-miR-590-5p MIMAT0003258GAGCUUAUucauaaaagugcag 535 hsa-miR-590-3p MIMAT0004801UAAUUUUAuguauaagcuagu 536 hsa-miR-591 MIMAT0003259 AGACCAUGgguucucauugu537 hsa-miR-592 MIMAT0003260 UUGUGUCAauaugcgaugaugu 538 hsa-miR-593*MIMAT0003261 AGGCACCAgccaggcauugcucagc 539 hsa-miR-593 MIMAT0004802UGUCUCUGcugggguuucu 540 hsa-miR-595 MIMAT0003263 GAAGUGUGccguggugugucu541 hsa-miR-596 MIMAT0003264 AAGCCUGCccggcuccucggg 542 hsa-miR-597MIMAT0003265 UGUGUCACucgaugaccacugu 543 hsa-miR-598 MIMAT0003266UACGUCAUcguugucaucguca 544 hsa-miR-599 MIMAT0003267 GUUGUGUCaguuuaucaaac545 hsa-miR-548a-5p MIMAT0004803 AAAAGUAAuugcgaguuuuacc 546 hsa-miR-600MIMAT0003268 ACUUACAGacaagagccuugcuc 547 hsa-miR-601 MIMAT0003269UGGUCUAGgauuguuggaggag 548 hsa-miR-602 MIMAT0003270GACACGGGcgacagcugcggccc 549 hsa-miR-603 MIMAT0003271CACACACUgcaauuacuuuugc 550 hsa-miR-604 MIMAT0003272 AGGCUGCGgaauucaggac551 hsa-miR-605 MIMAT0003273 UAAAUCCCauggugccuucuccu 552 hsa-miR-606MIMAT0003274 AAACUACUgaaaaucaaagau 553 hsa-miR-607 MIMAT0003275GUUCAAAUccagaucuauaac 554 hsa-miR-608 MIMAT0003276AGGGGUGGuguugggacagcuccgu 555 hsa-miR-609 MIMAT0003277AGGGUGUUucucucaucucu 556 hsa-miR-610 MIMAT0003278 UGAGCUAAaugugugcuggga557 hsa-miR-611 MIMAT0003279 GCGAGGACcccucggggucugac 558 hsa-miR-612MIMAT0003280 GCUGGGCAgggcuucugagcuccuu 559 hsa-miR-613 MIMAT0003281AGGAAUGUuccuucuuugcc 560 hsa-miR-614 MIMAT0003282GAACGCCUguucuugccaggugg 561 hsa-miR-615-5p MIMAT0004804GGGGGUCCccggugcucggauc 562 hsa-miR-615-3p MIMAT0003283UCCGAGCCugggucucccucuu 563 hsa-miR-616* MIMAT0003284ACUCAAAAcccuucagugacuu 564 hsa-miR-616 MIMAT0004805AGUCAUUGgaggguuugagcag 565 hsa-miR-548c-5p MIMAT0004806AAAAGUAAuugcgguuuuugcc 566 hsa-miR-548c-3p MIMAT0003285CAAAAAUCucaauuacuuuugc 567 hsa-miR-617 MIMAT0003286AGACUUCCcauuugaagguggc 568 hsa-miR-618 MIMAT0003287AAACUCUAcuuguccuucugagu 569 hsa-miR-619 MIMAT0003288GACCUGGAcauguuugugcccagu 570 hsa-miR-620 MIMAT0003289AUGGAGAUagauauagaaau 571 hsa-miR-621 MIMAT0003290 GGCUAGCAacagcgcuuaccu572 hsa-miR-622 MIMAT0003291 ACAGUCUGcugagguuggagc 573 hsa-miR-623MIMAT0003292 AUCCCUUGcaggggcuguugggu 574 hsa-miR-624* MIMAT0003293UAGUACCAguaccuuguguuca 575 hsa-miR-624 MIMAT0004807CACAAGGUauugguauuaccu 576 hsa-miR-625 MIMAT0003294 AGGGGGAAaguucuauagucc577 hsa-miR-625* MIMAT0004808 GACUAUAGaacuuucccccuca 578 hsa-miR-626MIMAT0003295 AGCUGUCUgaaaaugucuu 579 hsa-miR-627 MIMAT0003296GUGAGUCUcuaagaaaagagga 580 hsa-miR-628-5p MIMAT0004809AUGCUGACauauuuacuagagg 581 hsa-miR-628-3p MIMAT0003297UCUAGUAAgaguggcagucga 582 hsa-miR-629 MIMAT0004810 UGGGUUUAcguugggagaacu583 hsa-miR-629* MIMAT0003298 GUUCUCCCaacguaagcccagc 584 hsa-miR-630MIMAT0003299 AGUAUUCUguaccagggaaggu 585 hsa-miR-631 MIMAT0003300AGACCUGGcccagaccucagc 586 hsa-miR-33b MIMAT0003301 GUGCAUUGcuguugcauugc587 hsa-miR-33b* MIMAT0004811 CAGUGCCUcggcagugcagccc 588 hsa-miR-632MIMAT0003302 GUGUCUGCuuccuguggga 589 hsa-miR-633 MIMAT0003303CUAAUAGUaucuaccacaauaaa 590 hsa-miR-634 MIMAT0003304AACCAGCAccccaacuuuggac 591 hsa-miR-635 MIMAT0003305ACUUGGGCacugaaacaaugucc 592 hsa-miR-636 MIMAT0003306UGUGCUUGcucgucccgcccgca 593 hsa-miR-637 MIMAT0003307ACUGGGGGcuuucgggcucugcgu 594 hsa-miR-638 MIMAT0003308AGGGAUCGcgggcggguggcggccu 595 hsa-miR-639 MIMAT0003309AUCGCUGCgguugcgagcgcugu 596 hsa-miR-640 MIMAT0003310AUGAUCCAggaaccugccucu 597 hsa-miR-641 MIMAT0003311AAAGACAUaggauagagucaccuc 598 hsa-miR-642 MIMAT0003312GUCCCUCUccaaaugugucuug 599 hsa-miR-643 MIMAT0003313ACUUGUAUgcuagcucagguag 600 hsa-miR-644 MIMAT0003314 AGUGUGGCuuucuuagagc601 hsa-miR-645 MIMAT0003315 UCUAGGCUgguacugcuga 602 hsa-miR-646MIMAT0003316 AAGCAGCUgccucugaggc 603 hsa-miR-647 MIMAT0003317GUGGCUGCacucacuuccuuc 604 hsa-miR-648 MIMAT0003318 AAGUGUGCagggcacuggu605 hsa-miR-649 MIMAT0003319 AAACCUGUguuguucaagaguc 606 hsa-miR-650MIMAT0003320 AGGAGGCAgcgcucucaggac 607 hsa-miR-651 MIMAT0003321UUUAGGAUaagcuugacuuuug 608 hsa-miR-652 MIMAT0003322AAUGGCGCcacuaggguugug 609 hsa-miR-548d-5p MIMAT0004812AAAAGUAAuugugguuuuugcc 610 hsa-miR-548d-3p MIMAT0003323CAAAAACCacaguuucuuuugc 611 hsa-miR-661 MIMAT0003324UGCCUGGGucucuggccugcgcgu 612 hsa-miR-662 MIMAT0003325UCCCACGUuguggcccagcag 613 hsa-miR-663 MIMAT0003326AGGCGGGGcgccgcgggaccgc 614 hsa-miR-449b MIMAT0003327AGGCAGUGuauuguuagcuggc 615 hsa-miR-449b* MIMAT0009203CAGCCACAacuacccugccacu 616 hsa-miR-653 MIMAT0003328GUGUUGAAacaaucucuacug 617 hsa-miR-411 MIMAT0003329 UAGUAGACcguauagcguacg618 hsa-miR-411* MIMAT0004813 UAUGUAACacgguccacuaacc 619 hsa-miR-654-5pMIMAT0003330 UGGUGGGCcgcagaacaugugc 620 hsa-miR-654-3p MIMAT0004814UAUGUCUGcugaccaucaccuu 621 hsa-miR-655 MIMAT0003331AUAAUACAugguuaaccucuuu 622 hsa-miR-656 MIMAT0003332AAUAUUAUacagucaaccucu 623 hsa-miR-549 MIMAT0003333 UGACAACUauggaugagcucu624 hsa-miR-657 MIMAT0003335 GGCAGGUUcucacccucucuagg 625 hsa-miR-658MIMAT0003336 GGCGGAGGgaaguagguccguuggu 626 hsa-miR-659 MIMAT0003337CUUGGUUCagggagggucccca 627 hsa-miR-660 MIMAT0003338UACCCAUUgcauaucggaguug 628 hsa-miR-421 MIMAT0003339AUCAACAGacauuaauugggcgc 629 hsa-miR-542-5p MIMAT0003340UCGGGGAUcaucaugucacgaga 630 hsa-miR-542-3p MIMAT0003389UGUGACAGauugauaacugaaa 631 hsa-miR-758 MIMAT0003879UUUGUGACcugguccacuaacc 632 hsa-miR-1264 MIMAT0005791CAAGUCUUauuugagcaccuguu 633 hsa-miR-671-5p MIMAT0003880AGGAAGCCcuggaggggcuggag 634 hsa-miR-671-3p MIMAT0004819UCCGGUUCucagggcuccacc 635 hsa-miR-668 MIMAT0003881UGUCACUCggcucggcccacuac 636 hsa-miR-767-5p MIMAT0003882UGCACCAUgguugucugagcaug 637 hsa-miR-767-3p MIMAT0003883UCUGCUCAuaccccaugguuucu 638 hsa-miR-1224-5p MIMAT0005458GUGAGGACucgggaggugg 639 hsa-miR-1224-3p MIMAT0005459CCCCACCUccucucuccucag 640 hsa-miR-320b MIMAT0005792AAAAGCUGgguugagagggcaa 641 hsa-miR-320c MIMAT0005793AAAAGCUGgguugagagggu 642 hsa-miR-1296 MIMAT0005794UUAGGGCCcuggcuccaucucc 643 hsa-miR-1468 MIMAT0006789CUCCGUUUgccuguuucgcug 644 hsa-miR-1323 MIMAT0005795UCAAAACUgaggggcauuuucu 645 hsa-miR-1271 MIMAT0005796CUUGGCACcuagcaagcacuca 646 hsa-miR-1301 MIMAT0005797UUGCAGCUgccugggagugacuuc 647 hsa-miR-454* MIMAT0003884ACCCUAUCaauauugucucugc 648 hsa-miR-454 MIMAT0003885UAGUGCAAuauugcuuauagggu 649 hsa-miR-1185 MIMAT0005798AGAGGAUAcccuuuguauguu 650 hsa-miR-449c MIMAT0010251UAGGCAGUguauugcuagcggcugu 651 hsa-miR-449c* MIMAT0013771UUGCUAGUugcacuccucucugu 652 hsa-miR-1283 MIMAT0005799UCUACAAAggaaagcgcuuucu 653 hsa-miR-769-5p MIMAT0003886UGAGACCUcuggguucugagcu 654 hsa-miR-769-3p MIMAT0003887CUGGGAUCuccggggucuugguu 655 hsa-miR-766 MIMAT0003888ACUCCAGCcccacagccucagc 656 hsa-miR-762 MIMAT0010313GGGGCUGGggccggggccgagc 657 hsa-miR-802 MIMAT0004185CAGUAACAaagauucauccuugu 658 hsa-miR-670 MIMAT0010357GUCCCUGAguguauguggug 659 hsa-miR-1298 MIMAT0005800UUCAUUCGgcuguccagaugua 660 hsa-miR-2113 MIMAT0009206AUUUGUGCuuggcucugucac 661 hsa-miR-761 MIMAT0010364GCAGCAGGgugaaacugacaca 662 hsa-miR-764 MIMAT0010367GCAGGUGCucacuuguccuccu 663 hsa-miR-759 MIMAT0010497GCAGAGUGcaaacaauuuugac 664 hsa-miR-765 MIMAT0003945UGGAGGAGaaggaaggugaug 665 hsa-miR-770-5p MIMAT0003948UCCAGUACcacgugucagggcca 666 hsa-miR-675 MIMAT0004284UGGUGCGGagagggcccacagug 667 hsa-miR-675* MIMAT0006790CUGUAUGCccucaccgcuca 668 hsa-miR-298 MIMAT0004901AGCAGAAGcagggagguucuccca 669 hsa-miR-891a MIMAT0004902UGCAACGAaccugagccacuga 670 hsa-miR-300 MIMAT0004903UAUACAAGggcagacucucucu 671 hsa-miR-886-5p MIMAT0004905CGGGUCGGaguuagcucaagcgg 672 hsa-miR-886-3p MIMAT0004906CGCGGGUGcuuacugacccuu 673 hsa-miR-892a MIMAT0004907CACUGUGUccuuucugcguag 674 hsa-miR-220b MIMAT0004908CCACCACCgugucugacacuu 675 hsa-miR-450b-5p MIMAT0004909UUUUGCAAuauguuccugaaua 676 hsa-miR-450b-3p MIMAT0004910UUGGGAUCauuuugcauccaua 677 hsa-miR-874 MIMAT0004911CUGCCCUGgcccgagggaccga 678 hsa-miR-890 MIMAT0004912UACUUGGAaaggcaucaguug 679 hsa-miR-891b MIMAT0004913UGCAACUUaccugagucauuga 680 hsa-miR-220c MIMAT0004915ACACAGGGcuguugugaagacu 681 hsa-miR-888 MIMAT0004916UACUCAAAaagcugucaguca 682 hsa-miR-888* MIMAT0004917GACUGACAccucuuugggugaa 683 hsa-miR-892b MIMAT0004918CACUGGCUccuuucuggguaga 684 hsa-miR-541* MIMAT0004919AAAGGAUUcugcugucggucccacu 685 hsa-miR-541 MIMAT0004920UGGUGGGCacagaaucuggacu 686 hsa-miR-889 MIMAT0004921UUAAUAUCggacaaccauugu 687 hsa-miR-875-5p MIMAT0004922UAUACCUCaguuuuaucaggug 688 hsa-miR-875-3p MIMAT0004923CCUGGAAAcacugagguugug 689 hsa-miR-876-5p MIMAT0004924UGGAUUUCuuugugaaucacca 690 hsa-miR-876-3p MIMAT0004925UGGUGGUUuacaaaguaauuca 691 hsa-miR-708 MIMAT0004926AAGGAGCUuacaaucuagcuggg 692 hsa-miR-708* MIMAT0004927CAACUAGAcugugagcuucuag 693 hsa-miR-147b MIMAT0004928GUGUGCGGaaaugcuucugcua 694 hsa-miR-190b MIMAT0004929UGAUAUGUuugauauuggguu 695 hsa-miR-744 MIMAT0004945UGCGGGGCuagggcuaacagca 696 hsa-miR-744* MIMAT0004946CUGUUGCCacuaaccucaaccu 697 hsa-miR-885-5p MIMAT0004947UCCAUUACacuacccugccucu 698 hsa-miR-885-3p MIMAT0004948AGGCAGCGggguguaguggaua 699 hsa-miR-877 MIMAT0004949 GUAGAGGAgauggcgcaggg700 hsa-miR-877* MIMAT0004950 UCCUCUUCucccuccucccag 701 hsa-miR-887MIMAT0004951 GUGAACGGgcgccaucccgagg 702 hsa-miR-665 MIMAT0004952ACCAGGAGgcugaggccccu 703 hsa-miR-873 MIMAT0004953 GCAGGAACuugugagucuccu704 hsa-miR-543 MIMAT0004954 AAACAUUCgcggugcacuucuu 705 hsa-miR-374bMIMAT0004955 AUAUAAUAcaaccugcuaagug 706 hsa-miR-374b* MIMAT0004956CUUAGCAGguuguauuaucauu 707 hsa-miR-760 MIMAT0004957 CGGCUCUGggucugugggga708 hsa-miR-301b MIMAT0004958 CAGUGCAAugauauugucaaagc 709 hsa-miR-216bMIMAT0004959 AAAUCUCUgcaggcaaauguga 710 hsa-miR-208b MIMAT0004960AUAAGACGaacaaaagguuugu 711 hsa-miR-920 MIMAT0004970 GGGGAGCUguggaagcagua712 hsa-miR-921 MIMAT0004971 CUAGUGAGggacagaaccaggauuc 713 hsa-miR-922MIMAT0004972 GCAGCAGAgaauaggacuacguc 714 hsa-miR-924 MIMAT0004974AGAGUCUUgugaugucuugc 715 hsa-miR-509-3-5p MIMAT0004975UACUGCAGacguggcaaucaug 716 hsa-miR-933 MIMAT0004976UGUGCGCAgggagaccucuccc 717 hsa-miR-934 MIMAT0004977UGUCUACUacuggagacacugg 718 hsa-miR-935 MIMAT0004978CCAGUUACcgcuuccgcuaccgc 719 hsa-miR-936 MIMAT0004979ACAGUAGAgggaggaaucgcag 720 hsa-miR-937 MIMAT0004980AUCCGCGCucugacucucugcc 721 hsa-miR-938 MIMAT0004981UGCCCUUAaaggugaacccagu 722 hsa-miR-939 MIMAT0004982UGGGGAGCugaggcucugggggug 723 hsa-miR-940 MIMAT0004983AAGGCAGGgcccccgcucccc 724 hsa-miR-941 MIMAT0004984CACCCGGCugugugcacaugugc 725 hsa-miR-942 MIMAT0004985UCUUCUCUguuuuggccaugug 726 hsa-miR-943 MIMAT0004986CUGACUGUugccguccuccag 727 hsa-miR-944 MIMAT0004987AAAUUAUUguacaucggaugag 728 hsa-miR-297 MIMAT0004450AUGUAUGUgugcaugugcaug 729 hsa-miR-1178 MIMAT0005823UUGCUCACuguucuucccuag 730 hsa-miR-1179 MIMAT0005824AAGCAUUCuuucauugguugg 731 hsa-miR-1180 MIMAT0005825UUUCCGGCucgcgugggugugu 732 hsa-miR-1181 MIMAT0005826CCGUCGCCgccacccgagccg 733 hsa-miR-1182 MIMAT0005827GAGGGUCUugggagggaugugac 734 hsa-miR-1183 MIMAT0005828CACUGUAGgugauggugagagugggca 735 hsa-miR-1184 MIMAT0005829CCUGCAGCgacuugauggcuucc 736 hsa-miR-1225-5p MIMAT0005572GUGGGUACggcccagugggggg 737 hsa-miR-1225-3p MIMAT0005573UGAGCCCCugugccgcccccag 738 hsa-miR-1226* MIMAT0005576GUGAGGGCaugcaggccuggaugggg 739 hsa-miR-1226 MIMAT0005577UCACCAGCccuguguucccuag 740 hsa-miR-1227 MIMAT0005580CGUGCCACccuuuuccccag 741 hsa-miR-1228* MIMAT0005582GUGGGCGGgggcaggugugug 742 hsa-miR-1228 MIMAT0005583 UCACACCUgccucgcccccc743 hsa-miR-1229 MIMAT0005584 CUCUCACCacugcccucccacag 744 hsa-miR-1231MIMAT0005586 GUGUCUGGgcggacagcugc 745 hsa-miR-1233 MIMAT0005588UGAGCCCUguccucccgcag 746 hsa-miR-1234 MIMAT0005589UCGGCCUGaccacccaccccac 747 hsa-miR-1236 MIMAT0005591CCUCUUCCccuugucucuccag 748 hsa-miR-1237 MIMAT0005592UCCUUCUGcuccgucccccag 749 hsa-miR-1238 MIMAT0005593 CUUCCUCGucugucugcccc750 hsa-miR-1200 MIMAT0005863 CUCCUGAGccauucugagccuc 751 hsa-miR-1201MIMAT0005864 AGCCUGAUuaaacacaugcucuga 752 hsa-miR-1202 MIMAT0005865GUGCCAGCugcagugggggag 753 hsa-miR-1203 MIMAT0005866CCCGGAGCcaggaugcagcuc 754 hsa-miR-663b MIMAT0005867GGUGGCCCggccgugccugagg 755 hsa-miR-1204 MIMAT0005868UCGUGGCCuggucuccauuau 756 hsa-miR-1205 MIMAT0005869 UCUGCAGGguuugcuuugag757 hsa-miR-1206 MIMAT0005870 UGUUCAUGuagauguuuaagc 758 hsa-miR-1207-5pMIMAT0005871 UGGCAGGGaggcugggagggg 759 hsa-miR-1207-3p MIMAT0005872UCAGCUGGcccucauuuc 760 hsa-miR-1208 MIMAT0005873 UCACUGUUcagacaggcgga761 hsa-miR-548e MIMAT0005874 AAAAACUGagacuacuuuugca 762 hsa-miR-548jMIMAT0005875 AAAAGUAAuugcggucuuuggu 763 hsa-miR-1285 MIMAT0005876UCUGGGCAacaaagugagaccu 764 hsa-miR-1286 MIMAT0005877UGCAGGACcaagaugagcccu 765 hsa-miR-1287 MIMAT0005878UGCUGGAUcagugguucgaguc 766 hsa-miR-1289 MIMAT0005879UGGAGUCCaggaaucugcauuuu 767 hsa-miR-1290 MIMAT0005880UGGAUUUUuggaucaggga 768 hsa-miR-1291 MIMAT0005881UGGCCCUGacugaagaccagcagu 769 hsa-miR-548k MIMAT0005882AAAAGUACuugcggauuuugcu 770 hsa-miR-1293 MIMAT0005883UGGGUGGUcuggagauuugugc 771 hsa-miR-1294 MIMAT0005884UGUGAGGUuggcauuguugucu 772 hsa-miR-1295 MIMAT0005885UUAGGCCGcagaucuggguga 773 hsa-miR-1297 MIMAT0005886 UUCAAGUAauucaggug774 hsa-miR-1299 MIMAT0005887 UUCUGGAAuucugugugaggga 775 hsa-miR-5481MIMAT0005889 AAAAGUAUuugcggguuuuguc 776 hsa-miR-1302 MIMAT0005890UUGGGACAuacuuaugcuaaa 777 hsa-miR-1303 MIMAT0005891UUUAGAGAcggggucuugcucu 778 hsa-miR-1304 MIMAT0005892UUUGAGGCuacagugagaugug 779 hsa-miR-1305 MIMAT0005893UUUUCAACucuaaugggagaga 780 hsa-miR-1243 MIMAT0005894AACUGGAUcaauuauaggagug 781 hsa-miR-548f MIMAT0005895 AAAAACUGuaauuacuuuu782 hsa-miR-1244 MIMAT0005896 AAGUAGUUgguuuguaugagaugguu 783hsa-miR-1245 MIMAT0005897 AAGUGAUCuaaaggccuacau 784 hsa-miR-1246MIMAT0005898 AAUGGAUUuuuggagcagg 785 hsa-miR-1247 MIMAT0005899ACCCGUCCcguucguccccgga 786 hsa-miR-1248 MIMAT0005900ACCUUCUUguauaagcacugugcuaaa 787 hsa-miR-1249 MIMAT0005901ACGCCCUUcccccccuucuuca 788 hsa-miR-1250 MIMAT0005902ACGGUGCUggauguggccuuu 789 hsa-miR-1251 MIMAT0005903ACUCUAGCugccaaaggcgcu 790 hsa-miR-1253 MIMAT0005904AGAGAAGAagaucagccugca 791 hsa-miR-1254 MIMAT0005905AGCCUGGAagcuggagccugcagu 792 hsa-miR-1255a MIMAT0005906AGGAUGAGcaaagaaaguagauu 793 hsa-miR-1256 MIMAT0005907AGGCAUUGacuucucacuagcu 794 hsa-miR-1257 MIMAT0005908AGUGAAUGauggguucugacc 795 hsa-miR-1258 MIMAT0005909AGUUAGGAuuaggucguggaa 796 hsa-miR-1259 MIMAT0005910AUAUAUGAugacuuagcuuuu 797 hsa-miR-1260 MIMAT0005911 AUCCCACCucugccacca798 hsa-miR-548g MIMAT0005912 AAAACUGUaauuacuuuuguac 799 hsa-miR-1261MIMAT0005913 AUGGAUAAggcuuuggcuu 800 hsa-miR-1262 MIMAT0005914AUGGGUGAauuuguagaaggau 801 hsa-miR-1263 MIMAT0005915AUGGUACCcuggcauacugagu 802 hsa-miR-548n MIMAT0005916CAAAAGUAauuguggauuuugu 803 hsa-miR-548m MIMAT0005917CAAAGGUAuuugugguuuuug 804 hsa-miR-1265 MIMAT0005918CAGGAUGUggucaaguguuguu 805 hsa-miR-548o MIMAT0005919CCAAAACUgcaguuacuuuugc 806 hsa-miR-1266 MIMAT0005920CCUCAGGGcuguagaacagggcu 807 hsa-miR-1267 MIMAT0005921CCUGUUGAaguguaaucccca 808 hsa-miR-1268 MIMAT0005922 CGGGCGUGgugguggggg809 hsa-miR-1269 MIMAT0005923 CUGGACUGagccgugcuacugg 810 hsa-miR-1270MIMAT0005924 CUGGAGAUauggaagagcugugu 811 hsa-miR-1272 MIMAT0005925GAUGAUGAuggcagcaaauucugaaa 812 hsa-miR-1273 MIMAT0005926GGGCGACAaagcaagacucuuucuu 813 hsa-miR-1274a MIMAT0005927GUCCCUGUucaggcgcca 814 hsa-miR-548h MIMAT0005928 AAAAGUAAucgcgguuuuuguc815 hsa-miR-1275 MIMAT0005929 GUGGGGGAgaggcuguc 816 hsa-miR-1276MIMAT0005930 UAAAGAGCccuguggagaca 817 hsa-miR-302e MIMAT0005931UAAGUGCUuccaugcuu 818 hsa-miR-302f MIMAT0005932 UAAUUGCUuccauguuu 819hsa-miR-1277 MIMAT0005933 UACGUAGAuauauauguauuuu 820 hsa-miR-548pMIMAT0005934 UAGCAAAAacugcaguuacuuu 821 hsa-miR-548i MIMAT0005935AAAAGUAAuugcggauuuugcc 822 hsa-miR-1278 MIMAT0005936UAGUACUGugcauaucaucuau 823 hsa-miR-1279 MIMAT0005937 UCAUAUUGcuucuuucu824 hsa-miR-1274b MIMAT0005938 UCCCUGUUcgggcgcca 825 hsa-miR-1281MIMAT0005939 UCGCCUCCuccucuccc 826 hsa-miR-1282 MIMAT0005940UCGUUUGCcuuuuucugcuu 827 hsa-miR-1284 MIMAT0005941UCUAUACAgacccuggcuuuuc 828 hsa-miR-1288 MIMAT0005942UGGACUGCccugaucuggaga 829 hsa-miR-1292 MIMAT0005943UGGGAACGgguuccggcagacgcug 830 hsa-miR-1252 MIMAT0005944AGAAGGAAauugaauucauuua 831 hsa-miR-1255b MIMAT0005945CGGAUGAGcaaagaaagugguu 832 hsa-miR-1280 MIMAT0005946 UCCCACCGcugccaccc833 hsa-miR-1308 MIMAT0005947 GCAUGGGUgguucagugg 834 hsa-miR-664*MIMAT0005948 ACUGGCUAgggaaaaugauuggau 835 hsa-miR-664 MIMAT0005949UAUUCAUUuauccccagccuaca 836 hsa-miR-1306 MIMAT0005950 ACGUUGGCucugguggug837 hsa-miR-1307 MIMAT0005951 ACUCGGCGuggcgucggucgug 838 hsa-miR-513bMIMAT0005788 UUCACAAGgaggugucauuuau 839 hsa-miR-513c MIMAT0005789UUCUCAAGgaggugucguuuau 840 hsa-miR-1321 MIMAT0005952 CAGGGAGGugaaugugau841 hsa-miR-1322 MIMAT0005953 GAUGAUGCugcugaugcug 842 hsa-miR-720MIMAT0005954 UCUCGCUGgggccucca 843 hsa-miR-1197 MIMAT0005955UAGGACACauggucuacuucu 844 hsa-miR-1324 MIMAT0005956CCAGACAGaauucuaugcacuuuc 845 hsa-miR-1469 MIMAT0007347CUCGGCGCggggcgcgggcucc 846 hsa-miR-1470 MIMAT0007348GCCCUCCGcccgugcaccccg 847 hsa-miR-1471 MIMAT0007349GCCCGCGUguggagccaggugu 848 hsa-miR-1537 MIMAT0007399AAAACCGUcuaguuacaguugu 849 hsa-miR-1538 MIMAT0007400CGGCCCGGgcugcugcuguuccu 850 hsa-miR-1539 MIMAT0007401UCCUGCGCgucccagaugccc 851 hsa-miR-103-as MIMAT0007402UCAUAGCCcuguacaaugcugcu 852 hsa-miR-320d MIMAT0006764AAAAGCUGgguugagagga 853 hsa-miR-1825 MIMAT0006765 UCCAGUGCccuccucucc 854hsa-miR-1826 MIMAT0006766 AUUGAUCAucgacacuucgaacgcaau 855 hsa-miR-1827MIMAT0006767 UGAGGCAGuagauugaau 856 hsa-miR-1908 MIMAT0007881CGGCGGGGacggcgauugguc 857 hsa-miR-1909* MIMAT0007882UGAGUGCCggugccugcccug 858 hsa-miR-1909 MIMAT0007883CGCAGGGGccgggugcucaccg 859 hsa-miR-1910 MIMAT0007884CCAGUCCUgugccugccgccu 860 hsa-miR-1911 MIMAT0007885UGAGUACCgccaugucuguuggg 861 hsa-miR-1911* MIMAT0007886CACCAGGCauuguggucucc 862 hsa-miR-1912 MIMAT0007887UACCCAGAgcaugcagugugaa 863 hsa-miR-1913 MIMAT0007888UCUGCCCCcuccgcugcugcca 864 hsa-miR-1914 MIMAT0007889CCCUGUGCccggcccacuucug 865 hsa-miR-1914* MIMAT0007890GGAGGGGUcccgcacugggagg 866 hsa-miR-1915* MIMAT0007891ACCUUGCCuugcugcccgggcc 867 hsa-miR-1915 MIMAT0007892CCCCAGGGcgacgcggcggg 868 hsa-miR-1972 MIMAT0009447UCAGGCCAggcacaguggcuca 869 hsa-miR-1973 MIMAT0009448 ACCGUGCAaagguagcaua870 hsa-miR-1975 MIMAT0009450 CCCCCACAaccgcgcuugacuagcu 871 hsa-miR-1976MIMAT0009451 CCUCCUGCccuccuugcugu 872 hsa-miR-1979 MIMAT0009454CUCCCACUgcuucacuugacua 873 hsa-miR-2052 MIMAT0009977UGUUUUGAuaacaguaaugu 874 hsa-miR-2053 MIMAT0009978GUGUUAAUuaaaccucuauuuac 875 hsa-miR-2054 MIMAT0009979CUGUAAUAuaaauuuaauuuauu 876 hsa-miR-2110 MIMAT0010133UUGGGGAAacggccgcugagug 877 hsa-miR-2114 MIMAT0011156UAGUCCCUuccuugaagcgguc 878 hsa-miR-2114* MIMAT0011157CGAGCCUCaagcaagggacuu 879 hsa-miR-2115 MIMAT0011158AGCUUCCAugacuccugaugga 880 hsa-miR-2115* MIMAT0011159CAUCAGAAuucauggaggcuag 881 hsa-miR-2116 MIMAT0011160GGUUCUUAgcauaggaggucu 882 hsa-miR-2116* MIMAT0011161CCUCCCAUgccaagaacuccc 883 hsa-miR-2117 MIMAT0011162UGUUCUCUuugccaaggacag 884 hsa-miR-548q MIMAT0011163GCUGGUGCaaaaguaauggcgg 885 hsa-miR-2276 MIMAT0011775UCUGCAAGugucagaggcgagg 886 hsa-miR-2277 MIMAT0011777UGACAGCGcccugccuggcuc 887 hsa-miR-2278 MIMAT0011778GAGAGCAGuguguguugccugg 888 hsa-miR-711 MIMAT0012734GGGACCCAgggagagacguaag 889 hsa-miR-718 MIMAT0012735CUUCCGCCccgccgggcgucg 890 hsa-miR-2861 MIMAT0013802 GGGGCCUGgcggugggcgg891 hsa-miR-2909 MIMAT0013863 GUUAGGGCcaacaucucuugg 892 hsa-miR-3115MIMAT0014977 AUAUGGGUuuacuaguuggu 893 hsa-miR-3116 MIMAT0014978UGCCUGGAacauaguagggacu 894 hsa-miR-3117 MIMAT0014979AUAGGACUcauauagugccag 895 hsa-miR-3118 MIMAT0014980UGUGACUGcauuaugaaaauucu 896 hsa-miR-3119 MIMAT0014981UGGCUUUUaacuuugauggc 897 hsa-miR-3120 MIMAT0014982 CACAGCAAguguagacaggca898 hsa-miR-3121 MIMAT0014983 UAAAUAGAguaggcaaaggaca 899 hsa-miR-3122MIMAT0014984 GUUGGGACaagaggacggucuu 900 hsa-miR-3123 MIMAT0014985CAGAGAAUuguuuaauc 901 hsa-miR-3124 MIMAT0014986 UUCGCGGGcgaaggcaaaguc902 hsa-miR-548s MIMAT0014987 AUGGCCAAaacugcaguuauuuu 903 hsa-miR-3125MIMAT0014988 UAGAGGAAgcuguggagaga 904 hsa-miR-3126-5p MIMAT0014989UGAGGGACagaugccagaagca 905 hsa-miR-3126-3p MIMAT0015377CAUCUGGCauccgucacacaga 906 hsa-miR-3127 MIMAT0014990AUCAGGGCuuguggaaugggaag 907 hsa-miR-3128 MIMAT0014991UCUGGCAAguaaaaaacucucau 908 hsa-miR-3129 MIMAT0014992GCAGUAGUguagagauugguuu 909 hsa-miR-3130-5p MIMAT0014995UACCCAGUcuccggugcagcc 910 hsa-miR-3130-3p MIMAT0014994GCUGCACCggagacuggguaa 911 hsa-miR-3131 MIMAT0014996UCGAGGACugguggaagggccuu 912 hsa-miR-3132 MIMAT0014997UGGGUAGAgaaggagcucagagga 913 hsa-miR-3133 MIMAT0014998UAAAGAACucuuaaaacccaau 914 hsa-miR-378b MIMAT0014999 ACUGGACUuggaggcagaa915 hsa-miR-3134 MIMAT0015000 UGAUGGAUaaaagacuacauauu 916 hsa-miR-3135MIMAT0015001 UGCCUAGGcugagacugcagug 917 hsa-miR-466 MIMAT0015002AUACACAUacacgcaacacacau 918 hsa-miR-3136 MIMAT0015003CUGACUGAauagguagggucauu 919 hsa-miR-544b MIMAT0015004ACCUGAGGuugugcauuucuaa 920 hsa-miR-3137 MIMAT0015005UCUGUAGCcugggagcaauggggu 921 hsa-miR-3138 MIMAT0015006UGUGGACAgugagguagagggagu 922 hsa-miR-3139 MIMAT0015007UAGGAGCUcaacagaugccuguu 923 hsa-miR-3140 MIMAT0015008AGCUUUUGggaauucagguagu 924 hsa-miR-548t MIMAT0015009CAAAAGUGaucgugguuuuug 925 hsa-miR-3141 MIMAT0015010 GAGGGCGGguggaggagga926 hsa-miR-3142 MIMAT0015011 AAGGCCUUucugaaccuucaga 927 hsa-miR-3143MIMAT0015012 AUAACAUUguaaagcgcuucuuucg 928 hsa-miR-548u MIMAT0015013CAAAGACUgcaauuacuuuugcg 929 hsa-miR-3144-5p MIMAT0015014AGGGGACCaaagagauauauag 930 hsa-miR-3144-3p MIMAT0015015AUAUACCUguucggucucuuua 931 hsa-miR-3145 MIMAT0015016AGAUAUUUugaguguuuggaauug 932 hsa-miR-1273c MIMAT0015017GGCGACAAaacgagacccuguc 933 hsa-miR-3146 MIMAT0015018CAUGCUAGgauagaaagaaugg 934 hsa-miR-3147 MIMAT0015019GGUUGGGCagugaggaggguguga 935 hsa-miR-548v MIMAT0015020AGCUACAGuuacuuuugcacca 936 hsa-miR-3148 MIMAT0015021UGGAAAAAacuggugugugcuu 937 hsa-miR-3149 MIMAT0015022UUUGUAUGgauauguguguguau 938 hsa-miR-3150 MIMAT0015023CUGGGGAGauccucgagguugg 939 hsa-miR-3151 MIMAT0015024GGUGGGGCaaugggaucaggu 940 hsa-miR-3152 MIMAT0015025UGUGUUAGaauaggggcaauaa 941 hsa-miR-3153 MIMAT0015026GGGGAAAGcgaguagggacauuu 942 hsa-miR-3074 MIMAT0015027GAUAUCAGcucaguaggcaccg 943 hsa-miR-3154 MIMAT0015028CAGAAGGGgaguugggagcaga 944 hsa-miR-3155 MIMAT0015029CCAGGCUCugcagugggaacu 945 hsa-miR-3156 MIMAT0015030AAAGAUCUggaagugggagaca 946 hsa-miR-3157 MIMAT0015031UUCAGCCAggcuagugcagucu 947 hsa-miR-3158 MIMAT0015032AAGGGCUUccucucugcaggac 948 hsa-miR-3159 MIMAT0015033UAGGAUUAcaagugucggccac 949 hsa-miR-3160 MIMAT0015034AGAGCUGAgacuagaaagccca 950 hsa-miR-3161 MIMAT0015035CUGAUAAGaacagaggcccagau 951 hsa-miR-3162 MIMAT0015036UUAGGGAGuagaaggguggggag 952 hsa-miR-3163 MIMAT0015037UAUAAAAUgagggcaguaagac 953 hsa-miR-3164 MIMAT0015038UGUGACUUuaagggaaauggcg 954 hsa-miR-3165 MIMAT0015039AGGUGGAUgcaaugugaccuca 955 hsa-miR-3166 MIMAT0015040CGCAGACAaugccuacuggccua 956 hsa-miR-1260b MIMAT0015041AUCCCACCacugccaccau 957 hsa-miR-3167 MIMAT0015042 AGGAUUUCagaaauacuggugu958 hsa-miR-3168 MIMAT0015043 GAGUUCUAcagucagac 959 hsa-miR-3169MIMAT0015044 UAGGACUGugcuuggcacauag 960 hsa-miR-3170 MIMAT0015045CUGGGGUUcugagacagacagu 961 hsa-miR-3171 MIMAT0015046AGAUGUAUggaaucuguauauauc 962 hsa-miR-3172 MIMAT0015047UGGGGUUUugcaguccuua 963 hsa-miR-3173 MIMAT0015048 AAAGGAGGaaauaggcaggcca964 hsa-miR-1193 MIMAT0015049 GGGAUGGUagaccggugacgugc 965hsa-miR-323b-5p MIMAT0001630 AGGUUGUCcguggugaguucgca 966 hsa-miR-323b-3pMIMAT0015050 CCCAAUACacggucgaccucuu 967 hsa-miR-3174 MIMAT0015051UAGUGAGUuagagaugcagagcc 968 hsa-miR-3175 MIMAT0015052CGGGGAGAgaacgcagugacgu 969 hsa-miR-3176 MIMAT0015053 ACUGGCCUgggacuaccgg970 hsa-miR-3177 MIMAT0015054 UGCACGGCacuggggacacgu 971 hsa-miR-3178MIMAT0015055 GGGGCGCGgccggaucg 972 hsa-miR-3179 MIMAT0015056AGAAGGGGugaaauuuaaacgu 973 hsa-miR-3180-5p MIMAT0015057CUUCCAGAcgcuccgccccacgucg 974 hsa-miR-3180-3p MIMAT0015058UGGGGCGGagcuuccggaggcc 975 hsa-miR-548w MIMAT0015060AAAAGUAAcugcgguuuuugccu 976 hsa-miR-3181 MIMAT0015061AUCGGGCCcucggcgccgg 977 hsa-miR-3182 MIMAT0015062 GCUUCUGUaguguaguc 978hsa-miR-3183 MIMAT0015063 GCCUCUCUcggagucgcucgga 979 hsa-miR-3184MIMAT0015064 UGAGGGGCcucagaccgagcuuuu 980 hsa-miR-3185 MIMAT0015065AGAAGAAGgcggucggucugcgg 981 hsa-miR-3065-5p MIMAT0015066UCAACAAAaucacugaugcugga 982 hsa-miR-3065-3p MIMAT0015378UCAGCACCaggauauuguuggag 983 hsa-miR-3186-5p MIMAT0015067CAGGCGUCugucuacguggcuu 984 hsa-miR-3186-3p MIMAT0015068UCACGCGGagagauggcuuug 985 hsa-miR-3187 MIMAT0015069 UUGGCCAUggggcugcgcgg986 hsa-miR-3188 MIMAT0015070 AGAGGCUUugugcggauacgggg 987 hsa-miR-3189MIMAT0015071 CCCUUGGGucugaugggguag 988 hsa-miR-320e MIMAT0015072AAAGCUGGguugagaagg 989 hsa-miR-3190-5p MIMAT0015073UGUGGAAGguagacggccagaga 990 hsa-miR-3190-3p MIMAT0015074UGGAAGGUagacggccagagag 991 hsa-miR-3191 MIMAT0015075UGGGGACGuagcuggccagacag 992 hsa-miR-3192 MIMAT0015076UCUGGGAGguuguagcaguggaa 993 hsa-miR-3193 MIMAT0015077UCCUGCGUaggaucugaggagu 994 hsa-miR-3194 MIMAT0015078GGCCAGCCaccaggagggcug 995 hsa-miR-3195 MIMAT0015079 CGCGCCGGgcccggguu996 hsa-miR-3196 MIMAT0015080 CGGGGCGGcaggggccuc 997 hsa-miR-548xMIMAT0015081 UAAAAACUgcaauuacuuuca 998 hsa-miR-3197 MIMAT0015082GGAGGCGCaggcucggaaaggcg 999 hsa-miR-3198 MIMAT0015083GUGGAGUCcuggggaauggaga 1000 hsa-miR-3199 MIMAT0015084AGGGACUGccuuaggagaaaguu 1001 hsa-miR-3200 MIMAT0015085CACCUUGCgcuacucaggucug 1002 hsa-miR-3201 MIMAT0015086 GGGAUAUGaagaaaaau1003 hsa-miR-514b-5p MIMAT0015087 UUCUCAAGagggaggcaaucau 1004hsa-miR-514b-3p MIMAT0015088 AUUGACACcucugugagugga 1005 hsa-miR-3202MIMAT0015089 UGGAAGGGagaagagcuuuaau 1006 hsa-miR-1273d MIMAT0015090GAACCCAUgagguugaggcugcagu 1007 hsa-miR-4295 MIMAT0016844CAGUGCAAuguuuuccuu 1008 hsa-miR-4296 MIMAT0016845 AUGUGGGCucaggcuca 1009hsa-miR-4297 MIMAT0016846 UGCCUUCCugucugug 1010 hsa-miR-378cMIMAT0016847 ACUGGACUuggagucagaagagugg 1011 hsa-miR-4293 MIMAT0016848CAGCCUGAcaggaacag 1012 hsa-miR-4294 MIMAT0016849 GGGAGUCUacagcaggg 1013hsa-miR-4301 MIMAT0016850 UCCCACUAcuucacuuguga 1014 hsa-miR-4299MIMAT0016851 GCUGGUGAcaugagaggc 1015 hsa-miR-4298 MIMAT0016852CUGGGACAggaggaggaggcag 1016 hsa-miR-4300 MIMAT0016853 UGGGAGCUggacuacuuc1017 hsa-miR-4304 MIMAT0016854 CCGGCAUGuccagggca 1018 hsa-miR-4302MIMAT0016855 CCAGUGUGgcucagcgag 1019 hsa-miR-4303 MIMAT0016856UUCUGAGCugaggacag 1020 hsa-miR-4305 MIMAT0016857 CCUAGACAccuccaguuc 1021hsa-miR-4306 MIMAT0016858 UGGAGAGAaaggcagua 1022 hsa-miR-4309MIMAT0016859 CUGGAGUCuaggauucca 1023 hsa-miR-4307 MIMAT0016860AAUGUUUUuuccuguuucc 1024 hsa-miR-4308 MIMAT0016861 UCCCUGGAguuucuucuu1025 hsa-miR-4310 MIMAT0016862 GCAGCAUUcauguccc 1026 hsa-miR-4311MIMAT0016863 GAAAGAGAgcugagugug 1027 hsa-miR-4312 MIMAT0016864GGCCUUGUuccugucccca 1028 hsa-miR-4313 MIMAT0016865 AGCCCCCUggccccaaaccc1029 hsa-miR-4315 MIMAT0016866 CCGCUUUCugagcuggac 1030 hsa-miR-4316MIMAT0016867 GGUGAGGCuagcuggug 1031 hsa-miR-4314 MIMAT0016868CUCUGGGAaaugggacag 1032 hsa-miR-4318 MIMAT0016869 CACUGUGGguacaugcu 1033hsa-miR-4319 MIMAT0016870 UCCCUGAGcaaagccac 1034 hsa-miR-4320MIMAT0016871 GGGAUUCUguagcuuccu 1035 hsa-miR-4317 MIMAT0016872ACAUUGCCagggaguuu 1036 hsa-miR-4322 MIMAT0016873 CUGUGGGCucagcgcgugggg1037 hsa-miR-4321 MIMAT0016874 UUAGCGGUggaccgcccugcg 1038 hsa-miR-4323MIMAT0016875 CAGCCCCAcagccucaga 1039 hsa-miR-4324 MIMAT0016876CCCUGAGAcccuaaccuuaa 1040 hsa-miR-4256 MIMAT0016877 AUCUGACCugaugaaggu1041 hsa-miR-4257 MIMAT0016878 CCAGAGGUggggacugag 1042 hsa-miR-4258MIMAT0016879 CCCCGCCAccgccuugg 1043 hsa-miR-4259 MIMAT0016880CAGUUGGGucuaggggucagga 1044 hsa-miR-4260 MIMAT0016881CUUGGGGCauggaguccca 1045 hsa-miR-4253 MIMAT0016882 AGGGCAUGuccagggggu1046 hsa-miR-4251 MIMAT0016883 CCUGAGAAaagggccaa 1047 hsa-miR-4254MIMAT0016884 GCCUGGAGcuacuccaccaucuc 1048 hsa-miR-4255 MIMAT0016885CAGUGUUCagagaugga 1049 hsa-miR-4252 MIMAT0016886 GGCCACUGagucagcacca1050 hsa-miR-4325 MIMAT0016887 UUGCACUUgucucaguga 1051 hsa-miR-4326MIMAT0016888 UGUUCCUCugucucccagac 1052 hsa-miR-4327 MIMAT0016889GGCUUGCAugggggacugg 1053 hsa-miR-4261 MIMAT0016890 AGGAAACAgggaccca 1054hsa-miR-4265 MIMAT0016891 CUGUGGGCucagcucuggg 1055 hsa-miR-4266MIMAT0016892 CUAGGAGGccuuggcc 1056 hsa-miR-4267 MIMAT0016893UCCAGCUCgguggcac 1057 hsa-miR-4262 MIMAT0016894 GACAUUCAgacuaccug 1058hsa-miR-2355 MIMAT0016895 AUCCCCAGauacaauggacaa 1059 hsa-miR-4268MIMAT0016896 GGCUCCUCcucucaggaugug 1060 hsa-miR-4269 MIMAT0016897GCAGGCACagacagcccuggc 1061 hsa-miR-4263 MIMAT0016898 AUUCUAAGugccuuggcc1062 hsa-miR-4264 MIMAT0016899 ACUCAGUCauggucauu 1063 hsa-miR-4270MIMAT0016900 UCAGGGAGucaggggagggc 1064 hsa-miR-4271 MIMAT0016901GGGGGAAGaaaaggugggg 1065 hsa-miR-4272 MIMAT0016902 CAUUCAACuagugauugu1066 hsa-miR-4273 MIMAT0016903 GUGUUCUCugauggacag 1067 hsa-miR-4276MIMAT0016904 CUCAGUGAcucaugugc 1068 hsa-miR-4275 MIMAT0016905CCAAUUACcacuucuuu 1069 hsa-miR-4274 MIMAT0016906 CAGCAGUCccucccccug 1070hsa-miR-4281 MIMAT0016907 GGGUCCCGgggagggggg 1071 hsa-miR-4277MIMAT0016908 GCAGUUCUgagcacaguacac 1072 hsa-miR-4279 MIMAT0016909CUCUCCUCccggcuuc 1073 hsa-miR-4278 MIMAT0016910 CUAGGGGGuuugcccuug 1074hsa-miR-4280 MIMAT0016911 GAGUGUAGuucugagcagagc 1075 hsa-miR-4282MIMAT0016912 UAAAAUUUgcauccagga 1076 hsa-miR-4285 MIMAT0016913GCGGCGAGuccgacucau 1077 hsa-miR-4283 MIMAT0016914 UGGGGCUCagcgaguuu 1078hsa-miR-4284 MIMAT0016915 GGGCUCACaucaccccau 1079 hsa-miR-4286MIMAT0016916 ACCCCACUccugguacc 1080 hsa-miR-4287 MIMAT0016917UCUCCCUUgagggcacuuu 1081 hsa-miR-4288 MIMAT0016918 UUGUCUGCugaguuucc1082 hsa-miR-4292 MIMAT0016919 CCCCUGGGccggccuugg 1083 hsa-miR-4289MIMAT0016920 GCAUUGUGcagggcuauca 1084 hsa-miR-4290 MIMAT0016921UGCCCUCCuuucuucccuc 1085 hsa-miR-4291 MIMAT0016922 UUCAGCAGgaacagcu 1086hsa-miR-4329 MIMAT0016923 CCUGAGACccuaguuccac 1087 hsa-miR-4330MIMAT0016924 CCUCAGAUcagagccuugc 1088 hsa-miR-500b MIMAT0016925AAUCCUUGcuaccugggu 1089 hsa-miR-4328 MIMAT0016926 CCAGUUUUcccaggauu 1090

EXAMPLES Example 1

Segmented miRNA Mimetics

MicroRNAs (miRNAs) are a class of ˜22 nt noncoding RNAs that playimportant roles in regulating gene expression in plants and animals.MicroRNAs are usually produced by a process in which a RNA pol IItranscript is cut by Drosha to produce a precursor hairpin, which is cutby Dicer in the cytoplasm to produce a two stranded duplex that isincorporated into Argonaute (Ago) proteins. After elimination of thepassenger strand by cleavage or helicase activity, the guide strand canthen bind to complementary target RNAs. Studies have found that the mostprevalent aspect of miRNA target recognition is complementary binding toa target 3′ UTR by the miRNA seed region (positions 2 through 8 at the5′ end of the guide strand), leading to downregulation at mRNA andprotein levels.

Ago2 mediated cleavage of the passenger strand has been found to beimportant for assembly of siRNAs and some miRNAs and a nicked passengerstrand was found to rescue the activity of an siRNA containing aphosphorothioate bond that prevented passenger strand cleavage by Ago.This concept has been applied to the design of siRNAs (Bramsen et al.,2007 supra), where passenger segmentation was found to maintain siRNAactivity (while eliminating passenger strand activity), while guidesegmentation was found to eliminate the desired siRNA activity.

Applicant has surprisingly found that, as opposed to siRNA, segmentationis well tolerated in the guide strand of microRNA. Applicantdemonstrates herein that this segmentation provides for the alternativedesign of various miRNA mimetics that include nicks and gaps, as well assubstitutions and insertions that can confer additional propertiestoward therapeutic use.

Materials and Methods

Oligonucleotides used to obtain data in this Example were synthesized atSigma-Aldrich or Merck & Co. using standard methodologies. Annealing wasaccomplished by mixing single stranded RNA at 10 uM in 10 mM TrisHCl/50mM NaCl and heating at 95° C. for 2 minutes before slowly cooling to 37°C. over the course of 1 hour.

The RNA oligonucleotides synthesized are shown in the following TableII.

TABLE II SEQ Name Sequence ID NO. G UUAAGGCACGCGGUGAAUGCCA 1091 PGCAUUCACCGCGUGCCUUAAAU 1092 G10 UUAAGGCACG 1093 G.10 CGGUGAAUGCCA 1094G.9 GGUGAAUGCCA 1095 P10 GCAUUCACCG 1096 P.10 CGUGCCUUAAAU 1097 G10iUUAAGGCACG-iB 1098 G10Cy3 UUAAGGCACG-Cy3 1099 58-merGCGUUCACCGCGGACCUUGAUUUAAAUGUCCAUA 1100 CAAUUAAGGCACGCGGUGAAUGCC 48-merGCGUUCACCGCGGACCUUGAUUUAAAUGUCCAUA 1101 CAAUUAAGGCACGCGGUGAAUGCC 10-merGGUGAAUGCC 1102

Sequences in the table above are shown in 5′ to 3′ orientation. “iB”denotes an inverted abasic, while “Cy3” denotes a Cy3 fluorescent dyemolecule.

HCT-116 cells were cultured in McCoy's 5A Medium (Mediatech Inc.)supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin,plated in 96-well culture plates at a density of 25,000 cells/well 24hours prior to transfection, and then transfected using Opti-MEM IReduced Serum Media (Gibco) and Lipofectamine 2000 (Invitrogen) with afinal concentration of our miRNAs ranging from 30 nM down to 0.01 nMalong a 12-point titration curve. Twenty-four hours after transfection,cells were washed with phosphate-buffered saline and processed using theTaqMan® Gene Expression Cells-to-CT™ Kit (Applied Biosystems/Ambion) toextract RNA, synthesize cDNA, and perform RT-qPCR using CD164-specificprobes (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector.Reverse transcription conditions were as follows: 60 minutes at 37° C.followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used fordata normalization.

miRNAs were co-transfected with siCHECK2 vectors (Genscript) containingcloned target inserts, as shown in FIG. 14, consisting of a tandemrepeat of a seed match to miR-124 (2×7a), a seed match containingadditional 3′ complementarity to positions 13-17 of miR-124 (2×7a3p), ora full-length match to miR-124 (2×FL). A seed match with a two-basemutation (2×7aMutant) was used as a control. Twenty-four hours aftertransfection, transfection medium was replaced with fresh growth medium.Forty-eight hours after transfection, cells were lysed and both fireflyand Renilla luciferase activity were measured using the Dual-Glo™Luciferase Assay System (Promega) on a Wallac EnVision 2103 MultilabelReader (PerkinElmer).

HCT-116 cells were transfected with 10 nM miRNA duplex as describedpreviously (Jackson et al 2006). RNA was extracted using RNeasy(Qiagen), amplified using the Ovation protocol (Nugen), and profiled oncustom Affymetrix arrays (Rosetta Custom Human 1.0, Affymetrix). Arraysignals were analyzed with Affymetrix GeneChip Operating Software andAffymetrix Power Tools. UTR hexamer analysis was carried out asdescribed previously (Jackson et al 2006).

Results

Structural variants of a miR-124 duplex were tested, wherein a nick wasintroduced 10 nucleotides from the 5′ end of either the guide (or miRNA)or passenger (or miRNA*) strand (FIG. 12A). Duplexes were transfectedinto cells and changes in the mRNA levels of miR-124 target CD164 weremeasured. Division of the passenger strand (G/P10.10) had little effecton miR-124 activity (FIG. 12B), leading to a slight increase in EC50(0.12 nM to 0.29 nM). Division of the guide strand (G10.10/P) stillallowed for miR-124 activity (EC50 of 0.22 nM), indicating that acontinuous guide strand is not required for miRNA RISC activity. Theaddition of a one base gap between the guide halves, or capping of thejunction with a Cy3 dye or an inverted abasic residue, still gavemiR-124 activity, indicating that this activity was not a result ofligation of the guide halves. Toleration of guide strand segmentation isnot a property only of the miR-124 sequence, as division of the guidestrand in a miR-34 duplex still allowed for miR-34a activity (FIG. 9).

Microarrays were used to profile cells transfected with a miR-124 duplexcontaining the divided guide strand (FIG. 13A) to further confirm thetargeting activity of a segmented microRNA duplex, in this instance at agenome-wide level. Analysis of the 3′ UTR sequences of the downregulatedgenes shows that the most significantly enriched hexamer is GCCTTA,which corresponds to the seed sequence of miR-124. Profiling of theeffects of the segmented miR-124 duplex G10.10/P showed correlation(r=0.90) with profiling of the effects of the fully intact duplex (G/P),consistent with the preservation of the bulk of miR-124 targeting (FIG.13B).

Previous work analyzing microarray profiles has shown that although thepreponderance of miRNA targeting is due to seed sequence activity, amuch smaller degree of downregulation can be attributed to othercontributing factors, among them the supplementary binding of positions13-16 of the miRNA. Microarray profiling of miR-124 targets containingsupplementary 3′ binding was analyzed, and a shift following guidestrand segmentation was detected that was indicative of a loss ofsupplementary 3′ binding activity in the divided miRNA (FIG. 16).

The effects of segmented miRNAs on luciferase reporter vectors weretested, using miRNAs whose 3′ UTRs had been engineered (FIG. 14A) tocontain miRNA-complementary sites that constituted a full-length match(2×FL), a seed sequence match (2×7a), or a seed sequence plussupplementary 3′ match (2×7a3p). For an intact duplex (G/P), therepressive activity of miR-124 on luciferase activity was highest for2×FL and followed the order 2×FL-2×7a3p>2×7a. (FIG. 14B) Similarbehavior was seen with a segmented passenger strand (G/P10.10, FIG.14C). However, when the guide strand was divided (G10.10/P, FIG. 14D),the activities of the 2×FL and 2×7a3p reporters became equivalent tothat of 2×7a, showing that the discontinuity at position 10 of the guidestrand prevents productive 3′ binding, while permitting seed-basedactivity from the 5′ half.

Activity of a segmented guide strand was tested in the context of ahairpin sequence that was designed to emulate the natural miR-124hairpin. Appreciable activity was observed following guide stranddivision (FIG. 15B), indicating that processing of a hairpin into anAgo-recognizable duplex can occur in spite of a break in the guidestrand.

Example 2

Segmented miRNA Mimetics Targeted to CD164

MicroRNAs can down-regulate gene expression by inhibiting translation oftheir target transcripts and/or mediating the degradation of thesetranscripts. This Example demonstrates that certain of the segmentedmiRNA mimetic constructs according to the present disclosure designedbased on the corresponding naturally-occurring miRNAs are capable ofdoing the same. This example also indicates that segmented miRNAmimetics can comprise one or more locked nucleic acids (named “(L)”,underlined nucleotides are locked nucleic acid residues in Table III,IV, V and VI). Nicks are marked within the sequence as “(nick).” Gapsare marked within the sequence as “(€)” with each box indicating a onenucleotide gap.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimeticconstructs (sequences shown in Table III, passenger strand shown on topand guide strand on bottom) and a non-targeting control “UniversalControl (UC)” duplex were transfected into HCT116 DICER^(ex5), a humancolorectal cancer cell line with hypomorphic DICER function (Cummins, J.M., et al., PNAS 103:3687-3692, 2006). The transfections were carriedout using Lipofectamine RNAiMax (Invitrogen) per the manufacturer'sinstructions. RNA was isolated at 24 hours post-transfection using theRNeasy Kit (Qiagen) according to the manufacturer's instructions. Thetranscript abundance of the target gene, CD164, was measured by TaqmanReal-time PCR and Biomek NX (Biomek FX Dual-96).

Passenger strand sequences in Table III are shown in 5′ to 3′orientation and guide strand sequences are in 3′ to 5′ orientation.

TABLE III SEQ ID NOs. Name Sequence P G miR-(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 124/124(p)/124(g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)10(L).10(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1107/ 1091 (L)/(124g)ACCGUAAGUGGCGCACGGAAUU (guide) 1108 (124p)10.10/(124g)(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1096/ 1091ACCGUAAGUGGCGCACGGAAUU (guide) 1097 (124p)12(L).8(L)/(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU 1105/ 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) 1106 (124p)10(L).8(L)/(passenger) GCAUUCACCG(€€)UGCCUUAAAU 1107/ 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) 1109 (124p)/(124g)10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1103/ (L).10(L)ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1104 (124p)/(124a)10.10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1093/ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1094 (124p)/(124g)10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1103/ (L).9(L)ACCGUAAGUGG(€)GCACGGAAUU (guide) 1110 (124p)/(124g)10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1103/ (L).8(L)ACCGUAAGUG(€€)GCACGGAAUU (guide) 1112 (124p)/(124g)10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1103 (L).7(L)ACCGUAAGU(€€€)GCACGGAAUU (guide) (124p)/(124a)10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1093 (L).7ACCGUAAGU(€€€)GCACGGAAUU (guide) (124p)/(124g)11(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1111/ (L).9(L)ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1110 (124p)/(124g)11(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1111/ (L).8(L)ACCGUAAGUG(€)CGCACGGAAUU (guide) 1112 (124p)/(124g)11(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1111 (L).7(L)ACCGUAAGU(€€)CGCACGGAAUU (guide) (124p)/(124g)12(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1113/ (L).8(L)ACCGUAAGUG(nick)GCGCACGGAAUU (guide) 1112 (124p)/(124g)12(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1113 (L).7(L)ACCGUAAGU(€€)GCGCACGGAAUU (guide) (124p)/(124g)13(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1114 (L).7(L)ACCGUAAGU(nick)GGCGCACGGAAUU (guide)

Results of the activities are shown in FIG. 7. In brief, most of theconstructs tested demonstrated a capacity of knocking down CD164, aknown target to the naturally-occurring endogenous miR-124. Constructscomprising nicks in one or both strands of the segmented miRNA mimeticdemonstrated at least 25% of knockdown (e.g., 30%, 40%, 50%, 60%, 70%,80%, 90%, 100% or more) achieved by the non-segmented miR-24 positivecontrol.

Example 3

Segmented miRNA Mimetics Targeted to VAMP3

Segmented miRNA mimetics can be designed to include a discontinuitycomprising a nick or gap in one or both strands of any miRNA sequence ofthe invention in which target specific silencing activity is maintained.In the following example, nicks and gaps were introduced into miR-124miRNA mimetics and downstream target silencing was confirmed.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimeticconstructs (sequences shown in Table IV) and a non-targeting control“Universal Control (UC)” duplex were transfected into HCT116 DICERex5, ahuman colorectal cancer cell line with hypomorphic DICER function(Cummins, J. M., et al., PNAS 103:3687-3692, 2006). The transfectionswere carried out using Lipofectamine RNAiMax (Invitrogen) per themanufacturer's instructions. RNA was isolated at 24 hourspost-transfection using the RNeasy Kit (Qiagen) according to themanufacturer's instructions. The transcript abundance of the targetgene, VAMP3, was measured by Taqman Real-time PCR and Biomek NX (BiomekFX Dual-96).

Passenger strand sequences in Table IV are shown in 5′ to 3′ orientationand guide strand sequences are in 3′ to 5′ orientation.

TABLE IV SEQ ID NO(s). Name Sequence P G miR-(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 124/(124)/(124g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)10(L).10(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1107/ 1091 (L)/(124a)ACCGUAAGUGGCGCACGGAAUU (guide) 1108 (124p)/(124g)10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1103/ (L).10(L)ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1104 (124p)/(124g)11(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1111/ (L).9(L)ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1110 (124p)10(L).10(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1107/ 1103/ (L)/(124g)10(L).ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1108 1104 10(L) (124p)10(L).10(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1107/ 1093/ (L)/(124a)10.10ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1108 1094 (124p)10.10/(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1096/ 1103/ (124g)10(L).10ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1097 1104 (L) (124p)10.10/(124g)(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1096/ 1093/ 10.10ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1097 1094 (124p)10(L).10(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1107/ 1111/ (L)/(124g)11(L).ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1108 1110 9(L) (124p)10.10/(124g)(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1096/ 1111/ 11(L).9(L)ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1097 1110 (124p)10(L).10(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1107/ 1113/ (L)/(124g)12(L).ACCGUAAGUG(nick)GCGCACGGAAUU (guide) 1108 1112 8(L) (124p)10.10/(124g)(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1096/ 1113/ 12(L).8(L)ACCGUAAGUG(nick)GCGCACGGAAUU (guide) 1097 1112 (124p)10(L).10(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1107/ 1114 (L)/(124g)13(L).ACCGUAAGU(nick)GGCGCACGGAAUU (guide) 1108 7(L) (124p)10.10/(124g)(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1096/ 1114 13(L).7(L)ACCGUAAGU(nick)GGCGCACGGAAUU (guide) 1097 (124p)12(L).8(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU 1105/ 1103/ (L)/(124g)10(L).ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1106 1104 10(L) (124p)12(L).8(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU 1105/ 1103/ (L)/(124g)10.10ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1106 1104 (124p)12(L).8(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU 1105/ 1111/ (L)/(124g)11(L).ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1106 1110 9(L) (124p)12(L).8(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU 1105/ 1113/ (L)/(124g)12(L).ACCGUAAGUG(nick)GCGCACGGAAUU (guide) 1106 1112 8(L) (124p)12(L).8(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU 1105/ 1114 (L)/(124g)13(L).ACCGUAAGU(nick)GGCGCACGGAAUU (guide) 1106 7(L)

Results of this example are shown in FIG. 8. A number of the segmentedmiRNA mimetic constructs of this example, each strand comprising twodistinct contiguous stretches of nucleotides, achieved at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 100% of the knockdowneffect as compared to the non-segmented duplex miR-124 mimetic.

Example 4

Segmented miRNA Mimetics of miR-124 Versus miR-34

Segmented miRNA mimetics can be designed to include a discontinuitycomprising a nick or gap in any miRNA sequence of the invention in whichthe target specificity is maintained. In the following example, nicksand gaps were introduced into miR-124 and miR-34 miRNA mimetics anddownstream target specificity determined.

Synthetic duplex mimetic of miR-124 and segmented miR-124 constructs(sequences shown in Table V) and a non-targeting control “UniversalControl (UC)” duplex were transfected into HCT116 DICER^(ex5), a humancolorectal cancer cell line with hypomorphic DICER function (Cummins, J.M., et al., PNAS 103:3687-3692, 2006). The transfections were carriedout using Lipofectamine RNAiMax (Invitrogen) per the manufacturer'sinstructions. RNA was isolated at 24 hours post-transfection using theRNeasy Kit (Qiagen) according to the manufacturer's instructions. Thetranscript abundance of the target gene, CD164, was measured by TaqmanReal-time PCR and Biomek NX (Biomek FX Dual-96). The knockdown effectachieved by segmented miRNA-124 was also compared with the knockdown, orthe lack thereof, achieved by segmented miRNA-34 and a duplex miR-34mimetic, which are known to not target CD164.

Synthetic duplex mimetic of miR-34 and segmented miR-34 constructs(sequences shown in Table V) and a non-targeting control “UniversalControl (UC)” duplex were transfected into HCT116 DICER^(ex5), a humancolorectal cancer cell line with hypomorphic DICER function (Cummins, J.M., et al., PNAS 103:3687-3692, 2006). The transfections were carriedout using Lipofectamine RNAiMax (Invitrogen) per the manufacturer'sinstructions. RNA was isolated at 24 hours post-transfection using theRNeasy Kit (Qiagen) according to the manufacturer's instructions. Thetranscript abundance of the target gene, TK1, was measured by TaqmanReal-time PCR and Biomek NX (Biomek FX Dual-96). The knockdown effectachieved by segmented miRNA-34 was also compared with the knockdown, orthe lack thereof, achieved by segmented miRNA-124 and a duplex miR-124mimetic, which are known to not target TK1.

The nucleotides that were changed to effectuate the mismatches areindicated in lower case letters in the sequences. Passenger strandsequences in Table V are shown in 5′ to 3′ orientation and guide strandsequences are in 3′ to 5′ orientation.

TABLE V SEQ ID NO(s). Name Sequence P G miR-(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 124/(124p)/(124g)ACCGUAAGUGGCGCACGGAAUU (guide) miR-124 (blunt(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1115 end)CGUAAGUGGCGCACGGAAUU (guide) miR-124 (shorter(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1116 guide)UAAGUGGCGCACGGAAUU (guide) (124p)/(124a)10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1103/ (L).10(L)ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1104 (124p)/(124g)10.10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1093/ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1094 (124p)10.10/(124g)(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU 1096/ 1091ACCGUAAGUGGCGCACGGAAUU (guide) 1097 (124p)/(124g)10.10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1093/ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1094 (124p)/(124g)11.9(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1117/ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1118 (124p)/(124g)10.10m(passenger) GCAUcuACCG(nick)CGUGCCUUAAAU 1119/ 1093/ (mismatch)ACCGUAgaUGGC(nick)GCACGGAAUU (guide) 1097 1120 (124p)/(124g)10(passenger) GCAUUCACCG(nick)CGUcgCUUAAAU 1096/ 1122/ m3.10ACCGUAAGUGGC(nick)GCAgcGAAUU (guide) 1121 1094 (mismatch)(124p)/(124g)10 (passenger) GCAUUCACCG(nick)CGUGCCUauAAU 1096/ 1124/m5.10 ACCGUAAGUGG(nick)CGCACGGAuaU (guide) 1123 1118 (mismatch)(124p)/(124g)10.9 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1117/ACCGUAAGUG(€)CGCACGGAAUU (guide) 1125 (124p)/(124g)10(passenger) GCAUUCACCGCGUcgCUUAAAU 1126 1127 m3.9 (mismatch)ACCGUAAGU(€)GCGCAgcGAAUU (guide) (124p)/(124g)10(passenger) GCAUUCACCG(nick)CGUGCCUauAAU 1096/ 1128/ m5.9 (mismatch)ACCGUAAGUG(nick)GCGCACGGAuaU (guide) 1112 miR-(passenger) CACGAGCUAAGACACUGCUAAU 1093 1094 34a/(34p)/(34g)UGGUGCUCGAUUCUGUGACGGU (guide) (34p)/(34g)10.10(passenger) CACGAGCUAAGACACUGCUAAU 1093 1129/UGGUGCUCGAUU(nick)CUGUGACGGU (guide) 1130 (34p)/(34g)11.9(passenger) CACGAGCUAAGACACUGCUAAU 1093 1131/UGGUGCUCGAU(nick)UCUGUGACGGU (guide) 1132 (34p)/(34g)10.9(passenger) CACGAGCUAAGACACUGCUAAU 1093 1129/UGGUGCUCGAU(€)CUGUGACGGU (guide) 1132

The results are presented in FIGS. 9A and 9B. Certain segmented miRNAmimetic constructs of this example, achieved at least 20%, at least 25%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 100% of the knockdown effect ascompared to their respective non-segmented duplex mimetics.

Example 5

Segmented miRNA Mimetics Comprising Abasic Insertions

Segmented miRNA mimetics can be designed to include a discontinuitycomprising a nick or gap, in which one or more non-nucleotide moietiesof the invention are inserted into the terminal portions of sequenceadjacent to the nick or gap. In the following example, abasic moietieswere used to cap the internal ends of nucleotide positions in the guidestrand of a segmented miRNA mimetic. Likewise, insertions with othernon-nucleotide moieties described herein or otherwise known in the artcan be similarly performed by one of general skill following themethodologies herein.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimeticconstructs (sequences shown in Table VI), comprising one or moreinverted abasic modifications at the internal ends, and a non-targetingcontrol “Universal Control (UC)” duplex were transfected into HCT116DICER^(ex5), a human colorectal cancer cell line with hypomorphic DICERfunction (Cummins, J. M., et al., PNAS 103:3687-3692, 2006). Thetransfections were carried out using Lipofectamine RNAiMax (Invitrogen)per the manufacturer's instructions. RNA was isolated at 24 hourspost-transfection using the RNeasy Kit (Qiagen) according to themanufacturer's instructions. The transcript abundance of the targetgenes, CD164 and VAMP3, was measured by Taqman Real-time PCR and BiomekNX (Biomek FX Dual-96).

The position of the inverted abasic group is indicated as “(i)” in boththe names and the sequences of the following table. Passenger strandsequences in Table VI are shown in 5′ to 3′ orientation and guide strandsequences are in 3′ to 5′ orientation.

TABLE VI SEQ ID NO(s). Name Sequence P G miR-124(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091ACCGUAAGUGGCGCACGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1133 7(i).13(i)ACCGUAAGUGGCGCA(i)(nick)(i)CGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1134 8(i).12(i)ACCGUAAGUGGCGC(i)(nick)(i)ACGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1135 9(i).11(i)ACCGUAAGUGGCG(i)(nick)(i)CACGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1136/ 10(i).10(i)ACCGUAAGUGGC(i)(nick)(i)GCACGGAAUU (guide) 1137 (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1138/ 11(i).9(i)ACCGUAAGUGG(i)(nick)(i)CGCACGGAAUU (guide) 1139 (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1140/ 12(i).8(i)ACCGUAAGUG(i)(nick)(i)GCGCACGGAAUU (guide) 1141 (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1142 13(1).7(i)ACCGUAAGU(i)(nick)(i)GGCGCACGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1143 7(1).13ACCGUAAGUGGCGCA(nick)(i)CGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1144 8(i).12ACCGUAAGUGGCGC(nick)(i)ACGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1145 9(i).11ACCGUAAGUGGCG(nick)(i)CACGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1136/ 10(i).10ACCGUAAGUGGC(nick)(i)GCACGGAAUU (guide) 1094 (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1138/ 11(1).9ACCGUAAGUGG(nick)(i)CGCACGGAAUU (guide) 1118 (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1140/ 12(1).8ACCGUAAGUG(nick)(i)GCGCACGGAAUU (guide) 1125 (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1142 13(i).7ACCGUAAGU(nick)(i)GGCGCACGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1133 7.13(i)ACCGUAAGUGGCGCA(i)(nick)CGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1134 8.12(i)ACCGUAAGUGGCGC(i)(nick)ACGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1135 9.11(1)ACCGUAAGUGGCG(i)(nick)CACGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1093/ 10.10(1)ACCGUAAGUGGC(i)(nick)GCACGGAAUU (guide) 1137 (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1117/ 11.9(i)ACCGUAAGUGG(i)(nick)CGCACGGAAUU (guide) 1139 (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1146/ 12.8(i)ACCGUAAGUG(i)(nick)GCGCACGGAAUU (guide) 1141 (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1147 13.7(i)ACCGUAAGU(i)(nick)GGCGCACGGAAUU (guide)

The results of this example are indicated in FIGS. 10A and 10B. As canbe seen from this example, modifying the internal ends with one or moredeoxyabasic moieties or modifications can impart further improvement ofactivity to the segmented miRNA mimetics of the invention.

Example 6

Segmented miRNA Mimetics Comprising Abasic Substitutions

Segmented miRNA mimetics can be designed to include a discontinuitycomprising one or more non-nucleotide substitutions of the inventionthat occupy deleted sequence portions. In the following example, abasiclinkers were used to substitute deleted nucleotide positions in both theguide and passenger strands of a miRNA mimetic. Likewise, substitutionwith other non-nucleotide linking moieties described herein or otherwiseknown in the art can be similarly performed by one of general skillfollowing the methodologies herein.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimeticconstructs (sequences shown in Table VII), comprising one or more abasicsubstitutions and a non-targeting control “Universal Control (UC)”duplex were transfected into HCT 116 DICER^(ex5), a human colorectalcancer cell line with hypomorphic DICER function (Cummins, J. M., etal., PNAS 103:3687-3692 (2006). The transfections were carried out usingLipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions.RNA was isolated at 24 hours post-transfection using the RNeasy Kit(Qiagen) according to the manufacturer's instructions. The transcriptabundance of the target genes, CD164 and VAMP3, was measured by TaqmanReal-time PCR and Biomek NX (Biomek FX Dual-96).

The positions of the abasic linker is indicated as “(ab)” in both thenames and the sequences of the following table. Passenger strandsequences in Table VII are shown in 5′ to 3′ orientation and guidestrand sequences are in 3′ to 5′ orientation.

TABLE VII SEQ ID NO(s). Name Sequence P G miR-124(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091ACCGUAAGUGGCGCACGG-AAUU (guide) (124p)8(ab)₂11/(passenger) GCAUUCAC(ab)(ab)CGUGCCUUAAAU 1148 1149 (124g)9(ab)₂12ACCGUAAGUGG(ab)(ab)CACGGAAUU (guide) (124p)7(ab)₃11/(passenger) GCAUUCA(ab)(ab)(ab)CGUGCCUUAAAU 1150 1151 (124g)9(ab)₃13ACCGUAAGUG(ab)(ab)(ab)CACGGAAUU (guide) (124p)/6(ab)₄11/(passenger) GCAUUC(ab)(ab)(ab)(ab)CGUGCCUUAAAU 1152 1153 (124g)9(ab)₄14ACCGUAAGU(ab)(ab)(ab)(ab)CACGGAAUU (guide) (124p)/5(ab)₅11/(passenger) GCAUU(ab)(ab)(ab)(ab)(ab)CGUGCCUUAAAU 1154 1155(124g)9(ab)₅15 ACCGUAAG(ab)(ab)(ab)(ab)(ab)CACGGAAUU (guide)(124p)/7(ab)₂10/ (passenger) GCAUUCA(ab)(ab)GCGUGCCUUAAAU 1148 1156(124g)10(ab)₂13 ACCGUAAGUG(ab)(ab)GCACGGAAUU (guide) (124p)/6(ab)₃10/(passenger) GCAUUC(ab)(ab)(ab)GCGUGCCUUAAAU 1157 1158 (124g)10(ab)₃14ACCGUAAGU(ab)(ab)(ab)GCACGGAAUU (guide) (124p)/5(ab)₄10/(passenger) GCAUU(ab)(ab)(ab)(ab)GCGUGCCUUAAAU 1159 1160 (124g)10(ab)₄15ACCGUAAG(ab)(ab)(ab)(ab)GCACGGAAUU (guide) (124p)/4(ab)₅10/(passenger) GCAU(ab)(ab)(ab)(ab)(ab)GCGUGCCUUAAAU 1161 1162(124g)10(ab)₅16 ACCGUAA(ab)(ab)(ab)(ab)(ab)GCACGGAAUU (guide)(124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1163 8(ab)₂11ACCGUAAGUGGC(ab)(ab)ACGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1164 8(ab)₃12ACCGUAAGUGG(ab)(ab)(ab)ACGGAAUU (guide) (124p)/(124g)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1165 8(ab)₄13ACCGUAAGUG(ab)(ab)(ab)(ab)ACGGAAUU (guide) (124p)9(ab)11/(passenger) GCAUUCACC(ab)CGUGCCUUAAAU 1166 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)8(ab)₂11/(passenger) GCAUUCAC(ab)(ab)CGUGCCUUAAAU 1148 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)7(ab)₃11/(passenger) GCAUUCA(ab)(ab)(ab)CGUGCCUUAAAU 1150 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)6(ab)₄11/(passenger) GCAUUC(ab)(ab)(ab)(ab)CGUGCCUUAAAU 1152 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)5(ab)₅11/(passenger) GCAUU(ab)(ab)(ab)(ab)(ab)CGUGCCUUAAAU 1154 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)4(ab)₆11/ (passenger) 1167 1091(124g) GCAU(ab)(ab)(ab)(ab)(ab)(ab)CGUGCCUUAAAUACCGUAAGUGGCGCACGGAAUU (guide) (124p)8(ab)10/(passenger) GCAUUCAC(ab)GCGUGCCUUAAAU 1168 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)7(ab)₂10/(passenger) GCAUUCA(ab)(ab)GCGUGCCUUAAAU 1169 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)6(ab)₃10/(passenger) GCAUUC(ab)(ab)(ab)GCGUGCCUUAAAU 1170 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)5(ab)₄10/(passenger) GCAUU(ab)(ab)(ab)(ab)GCGUGCCUUAAAU 1171 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)4(ab)₅10/(passenger) GCAU(ab)(ab)(ab)(ab)(ab)GCGUGCCUUAAAU 1172 1091 (124g)ACCGUAAGUGGCGCACGGAAUU (guide) (124p)3(ab)₆10/ (passenger) 1173 1091(124g) GCA(ab)(ab)(ab)(ab)(ab)(ab)GCGUGCCUUAAAUACCGUAAGUGGCGCACGGAAUU (guide) (124p)/(124g)10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1156 (ab)₂13ACCGUAAGUG(ab)(ab)GCACGGAAUU (guide) (124p)/(124g)10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1158 (ab)₃14ACCGUAAGU(ab)(ab)(ab)GCACGGAAUU (guide) (124p)/(124g)10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1160 (ab)₄15ACCGUAAG(ab)(ab)(ab)(ab)GCACGGAAUU (guide) (124p)/(124g)10(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1162 (ab)₅16ACCGUAA(ab)(ab)(ab)(ab)(ab)GCACGGAAUU (guide) (124p)/(124g)11(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1174 (ab)₂14ACCGUAAGU(ab)(ab)CGCACGGAAUU (guide) (124p)/(124g)11(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1175 (ab)₃15ACCGUAAG(ab)(ab)(ab)CGCACGGAAUU (guide) (124p)/(124g)11(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1176 (ab)₄16ACCGUAA(ab)(ab)(ab)(ab)CGCACGGAAUU (guide) (124p)/(124g)11(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1177 (ab)₅17ACCGUA(ab)(ab)(ab)(ab)(ab)CGCACGGAAUU (guide)

The results of this example are indicated in FIGS. 11A and 11B. As canbe seen from this example, abasic substitutions can impart furtheradvantageous properties to the segmented miRNA mimetics of theinvention.

Example 7

Segmented miRNA Mimetics with Multiple Nucleotide Position Deletions andSubstitutions

Segmented miRNA mimetics can be designed to include a discontinuitycomprising one or more non-nucleotide substitutions of the inventionthat occupy deleted sequence portions of 1 or more nucleotide positions.In the following example, alkyl linkers were used to substitute deletednucleotide positions in both the guide and passenger strands of a miRNAmimetic. Likewise, substitution with other non-nucleotide linkingmoieties described herein or otherwise known in the art can be similarlyperformed by one of general skill following the methodologies herein.

Oligonucleotides comprising C3 and C6 linkers were synthesized usingprotocols well known in the art (solid phase synthesis) usingcommercially available phosphoramidites, then purified by reversed phasesolid phase extraction (SPE). The C3 (C₃₃H₄₃N₂O₅P) and C6 (C₃₆H₄₉N₂O₅P)phosphoramidites were purchased from ChemGenes.

Briefly, the single strand oligonucleotides were synthesized usingphosphoramidite chemistry on an automated solid-phase synthesizer, usingprocedures as are generally known in the art (see for example U.S.application Ser. No. 12/064,014). A synthesis column was packed withsolid support derivatized with the first nucleoside residue (natural orchemically modified). Synthesis was initiated by detritylation of theacid labile 5′-O-dimethoxytrityl group to release the 5′-hydroxyl. Asuitably protected phosphoramidite and a suitable activator inacetonitrile were delivered simultaneously to the synthesis columnresulting in coupling of the amidite to the 5′-hydroxyl. The column wasthen washed with a solvent, such as acetonitrile. An oxidizing solution,such as an iodine solution was pumped through the column to oxidize thephosphite triester linkage P(III) to its phosphotriester P(V) analog.Unreacted 5′-hydroxyl groups were capped using reagents such as aceticanhydride in the presence of 2,6-lutidine and N-methylimidazole. Theelongation cycle was resumed with the detritylation step for the nextphosphoramidite incorporation. This process was repeated until thedesired sequence was synthesized. The synthesis concluded with the final5′-terminus protecting group (trityl or 5′-O-dimethoxytrityl).

Upon completion of the synthesis, the solid-support and associatedoligonucleotide were dried under argon pressure or vacuum. Aqueous basewas added and the mixture was heated to effect cleavage of the succinyllinkage, removal of the cyanoethyl phosphate protecting group, anddeprotection of the exocyclic amine protection.

The following process was performed on single strands that do notcontain ribonucleotides. After treating the solid support with theaqueous base, the mixture was filtered to separate the solid supportfrom the deprotected crude synthesis material. The solid support wasthen rinsed with DMSO, which is combined with the filtrate. Theresultant basic solution allows for retention of the5′-O-dimethoxytrityl group to remain on the 5′ terminal position(trityl-on).

For single strands that contain ribonucleotides, the following processwas performed. After treating the solid support with the aqueous base,the mixture was filtered to separate the solid support from thedeprotected crude synthesis material. The solid support was then rinsedwith dimethylsulfoxide (DMSO), which was combined with the filtrate.Fluoride reagent, such as triethylamine trihydrofluoride, was added tothe mixture, and the solution was heated. The reaction was quenched withsuitable buffer to provide a solution of crude single strand with the5′-O-dimethoxytrityl group on the final 5′ terminal position.

The trityl-on solution of each crude single strand was purified usingchromatographic purification, such as SPE RPC purification. Thehydrophobic nature of the trityl group permits stronger retention of thedesired full-length oligo than the non-tritylated truncated failuresequences. The failure sequences were selectively washed from the resinwith a suitable solvent, such as low percent acetonitrile. Retainedoligonucleotides were then detritylated on-column with trifluoroaceticacid to remove the acid-labile trityl group. Residual acid was washedfrom the column, a salt exchange was performed, and a final desalting ofthe material commenced. The full-length oligo was recovered in apurified form with an aqueous-organic solvent. The final product wasthen analyzed for purity (HPLC), identity (Maldi-TOF MS), and yield (UVA260). The oligos were dried via lyophilization or vacuum condensation.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimeticconstructs (sequences shown in Table VIII), from which bases have beendeleted, and a non-targeting control “Universal Control (UC3)” duplexwere transfected into HCT-116 cells (wild-type) and cultured in McCoy's5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and1% penicillin-streptomycin. These cells were plated in 96-well cultureplates at a density of 6000 cells/well 24 hours prior to transfection.Transfection was carried out using Opti-MEM I Reduced Serum Media(Gibco) and Lipofectamine RNAiMax (Invitrogen) with a finalconcentration of our miRNAs at 10 nM. Twenty-four hours aftertransfection, cells were washed with phosphate-buffered saline andprocessed using the TaqMan® Gene Expression Cells-to-CT™ Kit (AppliedBiosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCRusing gene-specific probes (Applied Biosystems) on an ABI Prism 7900HTSequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C.followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used fordata normalization. Knockdown of miR-124 targets was calculated as thetwo-fold change in target cDNA measured in experimentally-treated cellsrelative to the target cDNA measured in non-targeting control-treatedcells.

The positions of deleted bases are indicated as “(€)” in both the namesand the sequences of the following table. C3 and C6 linkers areidentified as “(c3)” and “(c6)”, respectively. Passenger strandsequences in Table VIII are shown in 5′ to 3′ orientation and guidestrand sequences are in 3′ to 5′ orientation.

TABLE VIII SEQ ID NO(s). Name Sequence P G miR-124 (iP/G)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091ACCGUAAGUGGCGCACGGAAUU (guide) iP9-10del/G11-(passenger) GCAUUCAC(€)(€)CGUGCCUUAAAU 1097 1093 12delACCGUAAGUG(€)(€)GCACGGAAUU (guide) iP8-10del/G11-(passenger) GCAUUCA(€)(€)(€)CGUGCCUUAAAU 1097 1093 13delACCGUAAGU(€)(€)(€)GCACGGAAUU (guide) iP7-10del/G11-(passenger) GCAUUC(€)(€)(€)(€)CGUGCCUUAAAU 1097 1093 14delACCGUAAG(€)(€)(€)(€)GCACGGAAUU (guide) iP7-11del/G10-(passenger) GCAUUC(€)(€)(€)(€)GUGCCUUAAAU 1178 n/a 14delACCGUAAG(€)(€)(€)(€)(€)CACGGAAUU (guide) iP7-12del/G9-(passenger) GCAUUC(€)(€)(€)(€)(€)(€)UGCCUUAAAU 1109 n/a 14delACCGUAAG(€)(€)(€)(€)(€)(€)ACGGAUU (guide) iP9-(passenger) GCAUUCAC(€)(€)(c3)CGUGCCUUAAAU 1179 1180 10_1x_c3/G11-ACCGUAAGUG(€)(€)(c3)GCACGGAAUU (guide) 12_1x_c3 iP8-(passenger) GCAUUCA(€)(€)(€)(c3)CGUGCCUUAAAU 1181 1182 10_1x_c3/G11-ACCGUAAGU(€)(€)(€)(c3)GCACGGAAUU (guide) 13_1x_c3 iP7-(passenger) GCAUUC(€)(€)(€)(€)(c3)CGUGCCUUAAAU 1183 1184 10_1x_c3/G11-ACCGUAAG(€)(€)(€)(€)(c3)GCACGGAAUU (guide) 14_1x_c3 iP7-(passenger) GCAUUC(€)(€)(€)(€)(€)(c3)GUGCCUUAAAU 1185 1186 11_1x_c3/G10-ACCGUAAG(€)(€)(€)(€)(€)(c3)CACGGAAAU (guide) 14_1x_c3 iP7-(passenger) GCAUUC(€)(€)(€)(€)(€)(€)(c3)UGCCUUAAAU 1187 118812_1x_c3/G9- ACCGUAAG(€)(€)(€)(€)(€)(€)(c3)ACGGAAUU (guide) 14_1x_c3iP9- (passenger) GCAUUCAC(€)(€)(c3)CGUGCCUUAAAU 1179 1189 10_1x_c3/G11-ACCGUAAGUG(€)(€)(c6)GCACGGAAUU (guide) 12_1x_c6 iP8-(passenger) GCAUUCA(€)(€)(€)(c3)CGUGCCUUAAAU 1181 1190 10_1x_c3/G11-ACCGUAAGU(€)(€)(€)(c6)GCACGGAAUU (guide) 13_1x_c6 iP7-(passenger) GCAUUC(€)(€)(€)(€)(c3)CGUGCCUUAAAU 1183 1191 10_1x_c3/G11-ACCGUAAG(€)(€)(€)(€)(c6)GCACGGAAUU (guide) 14_1x_c6 iP7-(passenger) GCAUUC(€)(€)(€)(€)(€)(c3)GUGCCUUAAAU 1185 1192 11_1x_c3/G10-ACCGUAAG(€)(€)(€)(€)(€)(c6)CACGGAAUU (guide) 14_1x_c6 iP7-(passenger) GCAUUC(€)(€)(€)(€)(€)(€)(c3)UGCCUUAAAU 1187 119311_1x_c3/G9- ACCGUAAG(€)(€)(€)(€)(€)(€)(c6)ACGGAAUU (guide) 14_1x_c6

The results of this example are shown in FIGS. 17A and 17B. A number ofthe segmented miRNA mimetic constructs of this example achieved asignificant knockdown effect as compared to the non-segmented duplexmiR-124.

Example 8

Segmented miRNA Mimetics Comprising Small Substitutions

Segmented miRNA mimetics can be designed to include a discontinuitycomprising non-nucleotide substitutions of the invention that occupydeleted nucleotide positions. In the following example, C3 alkyl linkerswere used to substitute deleted nucleotide positions in both the guideand passenger strand of a miRNA mimetic. Likewise, substitution withother non-nucleotide linking moieties described herein or otherwiseknown in the art can be similarly performed by one of general skillfollowing the methodologies herein.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimeticconstructs (sequences shown in Table IX), comprising C3 substitutions,and a non-targeting control “Universal Control (UC3)” duplex weretransfected into HCT-116 cells (wild-type) and cultured in McCoy's 5AMedium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1%penicillin-streptomycin. These cells were plated in 96-well cultureplates at a density of 6000 cells/well 24 hours prior to transfection.Transfection was carried out using Opti-MEM I Reduced Serum Media(Gibco) and Lipofectamine RNAiMax (Invitrogen) with a finalconcentration of our miRNAs at 10 nM. Twenty-four hours aftertransfection, cells were washed with phosphate-buffered saline andprocessed using the TaqMan® Gene Expression Cells-to-CT™ Kit (AppliedBiosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCRusing gene-specific probes (Applied Biosystems) on an ABI Prism 7900HTSequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C.followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used fordata normalization. Knockdown of miR-124 targets was calculated as thetwo-fold change in target cDNA measured in experimentally-treated cellsrelative to the target cDNA measured in non-targeting control-treatedcells.

The positions of c3 substitutions are shown in both the names and thesequences of the following table. C3 linkers are identified as “(c3)”.Passenger strand sequences in Table IX are shown in 5′ to 3′ orientationand guide strand sequences are in 3′ to 5′ orientation.

TABLE IX SEQ ID  NO(s). Name Sequence P G miR-124(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 (iP/G)ACCGUAAGUGGCGCACGGAAUU (guide) iP22-22c3/G(passenger) GCAUUCACCGCGUGCCUUAAA(c3) 1194 1091ACCGUAAGUGGCGCACGGAAUU (guide) iP21-21c3/G(passenger) GCAUUCACCGCGUGCCUUAA(c3)U 1195 1091ACCGUAAGUGGCGCACGGAAUU (guide) iP20-(passenger) GCAUUCACCGCGUGCCUUA(c3)AU 1196 1216 20c3/G1-1c3ACCGUAAGUGGCGCACGGAAU(c3) (guide) iP19-(passenger) GCAUUCACCGCGUGCCUU(c3)AAU 1197 1217 19c3/G2-2c3ACCGUAAGUGGCGCACGGAA(c3)U (guide) iP18-(passenger) GCAUUCACCGCGUGCCU(c3)AAAU 1198 1218 18c3/G3-3c3ACCGUAAGUGGCGCACGGA(c3)UU (guide) iP17-(passenger) GCAUUCACCGCGUCiCC(c3)UAAAU 1199 1219 17c3/G4-4c3ACCGUAAGUGGCGCACGG(c3)AUU (guide) iP16-(passenger) GCAUUCACCGCGUGC(c3)UUAAAU 1200 1220 16c3/G5-5c3ACCGUAAGUGGCGCACG(c3)AAUU (guide) iP15-(passenger) GCAUUCACCGCGUG(c3)CUUAAAU 1201 1221 15c3/G6-6c3ACCGUAAGUGGCGCAC(c3)GAAUU (guide) iP14-(passenger) GCAUUCACCGCGU(c3)CCUUAAAU 1202 1222 14c3/G7-7c3ACCGUAAGUGGCGCA(c3)GGAAUU (guide) iP13-(passenger) GCAUUCACCGCG(c3)GCCUUAAAU 1203 1223 13c3/G8-8c3ACCGUAAGUGGCGC(c3)CGGAAUU (guide) iP12-(passenger) GCAUUCACCGC(c3)UGCCUUAAAU 1204 1224 12c3/G9-9c3ACCGUAAGUGGCG(c3)ACGGAAUU (guide) iP11-(passenger) GCAUUCACCG(c3)GUGCCUUAAAU 1205 1225 11c3/G10-ACCGUAAGUGGC(c3)CACGGAAUU (guide) 10c3 iP10-(passenger) GCAUUCACC(c3)CGUGCCUUAAAU 1206 1226 10c3/G11-ACCGUAAGUGG(c3)GCACGGAAUU (guide) 11c3 iP9-9c3/G12-(passenger) GCAUUCAC(c3)GCGUGCCUUAAAU 1207 1227 12c3ACCGUAAGUG(c3)CGCACGGAAUU (guide) iP8-8c3/G13-(passenger) GCAUUCA(c3)CGCGUGCCUUAAAU 1208 1228 13c3ACCGUAAGU(c3)GCGCACGGAAUU (guide) iP7-7c3/G14-(passenger) GCAUUC(c3)CCGCGUGCCUUAAAU 1209 1229 14c3ACCGUAAG(c3)GGCGCACGGAAUU (guide) iP6-6c3/G15-(passenger) GCAUU(c3)ACCGCGUGCCUUAAAU 1210 1230 15c3ACCGUAA(c3)UGGCGCACGGAAUU (guide) iP5-5c3/G16-(passenger) GCAU(c3)CACCGCGUGCCUUAAAU 1211 1231 16c3ACCGUA(c3)GUGGCGCACGGAAUU (guide) iP4-4c3/G17-(passenger) GCA(c3)UCACCGCGUGCCUUAAAU 1212 1232 17c3ACCGU(c3)AGUGGCGCACGGAAUU (guide) iP3-3c3/G18-(passenger) GC(c3)UUCACCGCGUGCCUUAAAU 1213 1233 18c3ACCG(c3)AAGUGGCGCACGGAAUU (guide) iP2-2c3/G19-(passenger) G(c3)AUUCACCGCGUGCCUUAAAU 1214 1234 19c3ACC(c3)UAAGUGGCGCACGGAAUU (guide) iP1-1c3/G20-(passenger) (c3)CAUUCACCGCGUGCCUUAAAU 1215 1235 20c3AC(c3)GUAAGUGGCGCACGGAAUU (guide) iP/G21-21c3(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1236A(c3)CGUAAGUGGCGCACGGAAUU (guide)

The results of this example are shown in FIGS. 18A and 18B. A number ofthe segmented miRNA mimetics of this example showed improved knockdownin comparison to the non-segmented duplex miR-124.

Example 9

Segmented miRNA Mimetics Comprising Larger Substitutions

Segmented miRNA mimetics can be designed to include a discontinuitycomprising non-nucleotide substitutions of the invention that occupydeleted nucleotide positions. In the following example, C6 alkyl linkerswere used to substitute deleted nucleotide positions in both the guideand passenger strand of a miRNA mimetic. Likewise, substitution withother larger non-nucleotide linking moieties described herein orotherwise known in the art can be similarly performed by one of generalskill following the methodologies herein.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimeticconstructs (sequences shown in Table X), comprising c6 substitutions,and a non-targeting control “Universal Control (UC3)” duplex weretransfected into HCT-116 cells (wild-type) and cultured in McCoy's 5AMedium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1%penicillin-streptomycin. These cells were plated in 96-well cultureplates at a density of 6000 cells/well 24 hours prior to transfection.Transfection was carried out using Opti-MEM I Reduced Serum Media(Gibco) and Lipofectamine RNAiMax (Invitrogen) with a finalconcentration of our miRNAs at 10 nM. Twenty-four hours aftertransfection, cells were washed with phosphate-buffered saline andprocessed using the TaqMan® Gene Expression Cells-to-CT™ Kit (AppliedBiosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCRusing gene-specific probes (Applied Biosystems) on an ABI Prism 7900HTSequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C.followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used fordata normalization. Knockdown of miR-124 targets was calculated as thetwo-fold change in target cDNA measured in experimentally-treated cellsrelative to the target cDNA measured in non-targeting control-treatedcells.

The positions of c6 substitutions are shown in both the names and thesequences of the following table. C6 linkers are identified as “(c6)”.Passenger strand sequences in Table X are shown in 5′ to 3′ orientationand guide strand sequences are in 3′ to 5′ orientation.

TABLE X SEQ ID  NO(s). Name Sequence P G miR-124(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 (iP/G)ACCGUAAGUGGCGCACGGAAUU (guide) iP22-22c6/G(passenger) GCAUUCACCGCGUGCCUUAAA(c6) 1237 1091ACCGUAAGUGGCGCACGGAAUU (guide) iP21-21c6/G(passenger) GCAUUCACCGCGUGCCUUAA(c6)U 1238 1091ACCGUAAGUGGCGCACGGAAUU (guide) iP20-(passenger) GCAUUCACCGCGUGCCUUA(c6)AU 1239 1259 20c6/G1-1c6ACCGUAAGUGGCGCACGGAAU(c6) (guide) iP19-(passenger) GCAUUCACCGCGUGCCUU(c6)AAU 1240 1260 19c6/G2-2c6ACCGUAAGUGGCGCACGGAA(c6)U (guide) iP18-(passenger) GCAUUCACCGCGUGCCU(c6)AAAU 1241 1261 18c6/G3-3c6ACCGUAAGUGGCGCACGGA(c6)UU (guide) iP17-(passenger) GCAUUCACCGCGUGCC(c6)UAAAU 1242 1262 17c6/G4-4c6ACCGUAAGUGGCGCACG-G(c6)AUU (guide) iP16-(passenger) GCAUUCACCGCGUGC(c6)UUAAAU 1243 1263 16c6/G5-5c6ACCGUAAGUGGCGCACG(c6)AAUU (guide) iP15-(passenger) GCAUUCACCGCGUG(c6)CUUAAAU 1244 1264 15c6/G6-6c6ACCGUAAGUGGCGCAC(c6)GAAUU (guide) iP14-(passenger) GCAUUCACCGCGU(c6)CCUUAAAU 1245 1265 14c6/G7-7c6ACCGUAAGUGGCGCA(c6)GGAAUU (guide) iP13-(passenger) GCAUUCACCGCG(c6)GCCUUAAAU 1246 1266 13c6/G8-8c6ACCGUAAGUGGCGC(c6)CGGAAUU (guide) iP12-(passenger) GCAUUCACCGC(c6)UGCCUUAAAU 1247 1267 12c6/G9-9c6ACCGUAAGUGGCG(c6)ACGGAAUU (guide) iP11-(passenger) GCAUUCACCG(c6)GUGCCUUAAAU 1248 1268 11c6/G10-ACCGUAAGUGGC(c6)CACGGAAUU (guide) 10c6 iP10-(passenger) GCAUUCACC(c6)CGUGCCUUAAAU 1249 1269 10c6/G11-ACCGUAAGUGG(c6)GCACGGAAUU (guide) 11c6 iP9-9c6/G12-(passenger) GCAUUCAC(c6)GCGUGCCUUAAAU 1250 1270 12c6ACCGUAAGUG(c6)CGCACGGAAUU (guide) iP8-8c6/G13-(passenger) GCAUUCA(c6)CGCGUGCCUUAAAU 1251 1271 13c6ACCGUAAGU(c6)GCGCACGGAAUU (guide) iP7-7c6/G14-(passenger) GCAUUC(c6)CCGCGUGCCUUAAAU 1252 1272 14c6ACCGUAAG(c6)GGCGCACGGAAUU (guide) iP6-6c6/G15-(passenger) GCAUU(c6)ACCGCGUGCCUUAAAU 1253 1273 15c6ACCGUAA(c6)UGGCGCACGGAAUU (guide) iP5-5c6/G16-(passenger) GCAU(c6)CACCGCGUGCCUUAAAU 1254 1274 16c6ACCGUA(c6)GUGGCGCACGGAAUU (guide) iP4-4c6/G17-(passenger) GCA(c6)UCACCGCGUGCCUUAAAU 1255 1275 17c6ACCGU(c6)AGUGGCGCACGGAAUU (guide) iP3-3c6/G18-(passenger) GC(c6)UUCACCGCGUGCCUUAAAU 1256 1276 18c6ACCG(c6)AAGUGGCGCACGGAAUU (guide) iP2-2c6/G19-(passenger) G(c6)AUUCACCGCGUGCCUUAAAU 1257 1277 19c6ACC(c6)UAAGUGGCGCACGGAAUU (guide) iP1-1c6/G20-(passenger) (c6)CAUUCACCGCGUGCCUUAAAU 1258 1278 20c6AC(c6)GUAAGUGGCGCACGGAAUU (guide) iP/G21-21c6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1279A(c6)CGUAAGUGGCGCACGGAAUU (guide)

The results of this example are indicated in FIGS. 19A and 19B. A numberof the segmented miRNA mimetics of this example showed increasedknockdown in comparison to the non-segmented duplex miR-124.

Example 10

Segmented miRNA Mimetics Comprising Non-Nucleotide Insertions

Segmented miRNA mimetics can be designed to include a discontinuitycomprising non-nucleotide insertions of the invention. In the followingexample, both small (C3) and larger (C6) alkyl moieties were used toconnect segmented positions in both the guide and passenger strand of amiRNA mimetic. Likewise, insertions with other non-nucleotide linkingmoieties described herein or otherwise known in the art can be similarlyperformed by one of general skill following the methodologies herein.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimeticconstructs (sequences shown in Table X1), comprising c3 and c6insertions, and a non-targeting control “Universal Control (UC3)” duplexwere transfected into HCT-116 cells (wild-type) and cultured in McCoy's5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and1% penicillin-streptomycin. These cells were plated in 96-well cultureplates at a density of 6000 cells/well 24 hours prior to transfection.Transfection was carried out using Opti-MEM I Reduced Serum Media(Gibco) and Lipofectamine RNAiMax (Invitrogen) with a finalconcentration of our miRNAs at 10 nM. Twenty-four hours aftertransfection, cells were washed with phosphate-buffered saline andprocessed using the TaqMan® Gene Expression Cells-to-CT™ Kit (AppliedBiosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCRusing gene-specific probes (Applied Biosystems) on an ABI Prism 7900HTSequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C.followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used fordata normalization. Knockdown of miR-124 targets was calculated as thetwo-fold change in target cDNA measured in experimentally-treated cellsrelative to the target cDNA measured in non-targeting control-treatedcells.

The positions of c3 and c6 insertions are shown in both the names andthe sequences of the following table. C3 and C6 linkers are identifiedas “(c3)” and “(c6)”, respectively. Passenger strand sequences in TableX1 are shown in 5′ to 3′ orientation and guide strand sequences are in3′ to 5′ orientation.

TABLE XI SEQ ID  NO(s). Name Sequence P G miR-124 (iP/G)(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091ACCGUAAGUGGCGCACGGAAUU (guide) iP/G1insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1280ACCGUAAGUGGCGCACGGAAU(c6)U (guide) iP/G2insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1281ACCGUAAGUGGCGCACGGAA(c6)UU (guide) iP/G3insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1282ACCGUAAGUGGCGCACGGA(c6)AUU (guide) iP/G4insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1283ACCGUAAGUGGCGCACGG(c6)AAUU (guide) iP/G5insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1284ACCGUAAGUGGCGCACG(c6)GAAUU (guide) iP/G6insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1285ACCGUAAGUGGCGCAC(c6)GGAAUU (guide) iP/G7insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1286ACCGUAAGUGGCGCA(c6)CGGAAUU (guide) iP/G8insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1287ACCGUAAGUGGCGC(c6)ACGGAAUU (guide) iP/G9insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1288ACCGUAAGUGGCG(c6)CACGGAAUU (guide) iP/G10insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1289ACCGUAAGUGGC(c6)GCACGGAAUU (guide) iP/G11insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1290ACCGUAAGUGG(c6)CGCACGGAAUU (guide) iP/G12insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1291ACCGUAAGUG(c6)GCGCACGGAAUU (guide) iP/G13insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1292ACCGUAAGU(c6)GGCGCACGGAAUU (guide) iP/G14insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1293ACCGUAAG(c6)UGGCGCACGGAAUU (guide) iP/G15insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1294ACCGUAA(c6)GUGGCGCACGGAAUU (guide) iP/G-16insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1295ACCGUA(c6)AGUGGCGCACGGAAUU (guide) iP/G17insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1296ACCGU(c6)AAGUGGCGCACGGAAUU (guide) iP/G18insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1297ACCG(c6)UAAGUGGCGCACGGAAUU (guide) iP/G19insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1298ACC(c6)GUAAGUGGCGCACGGAAUU (guide) iP/G20insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1299AC(c6)CGUAAGUGGCGCACGGAAUU (guide) iP/G21insertc6(passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1300A(c6)CCGUAAGUGGCGCACGGAAUU (guide) iP10insertc6/G(passenger) GCAUUCACCG(c6)CGUGCCUUAAAU 1301 1091ACCGUAAGUGGCGCACGGAAUU (guide) iP9insertc6/G(passenger) GCAUUCACC(c6)GCGUGCCUUAAAU 1302 1091ACCGUAAGUGGCGCACGGAAUU (guide) iP8insertc6/G(passenger) GCAUUCAC(c6)CGCGUGCCUUAAAU 1303 1091ACCGUAAGUGGCGCACGGAAUU (guide) iP7insertc6/G(passenger) GCAUUCA(c6)CCGCGUGCCUUAAAU 1304 1091ACCGUAAGUGGCGCACGGAAUU (guide) iP6insertc6/G(passenger) GCAUUC(c6)ACCGCGUGCCUUAAAU 1305 1091ACCGUAAGUGGCGCACGGAAUU (guide) iP10insertc6/G10insertc6(passenger) GCAUUCACCG(c6)CGUGCCUUAAAU 1301 1289ACCGUAAGUGGC(c6)GCACGGAAUU (guide) iP9insertc6/G11insertc6(passenger) GCAUUCAC(c6)CGCGUGCCUUAAAU 1302 1290ACCGUAAGUG(c6)GCGCACGGAAUU (guide) iP8insertc6/G12insertc6(passenger) GCAUUCA(c6)CCGCGUGCCUUAAAU 1303 1291ACCGUAAGU(c6)GGCGCACGGAAUU (guide) iP7insertc6/G13insertc6(passenger) GCAUUC(c6)ACCGCGUGCCUUAAAU 1304 1292ACCGUAAG(c6)UGGCGCACGGAAUU (guide) iP6insertc6/G14insertc6(passenger) GCAUU(c6)CACCGCGUGCCUUAAAU 1305 1293ACCGUAA(c6)GUGGCGCACGGAAUU (guide) iP10insertc3/G10insertc3(passenger) GCAUUCACCG(c3)CGUGCCUUAAAU 1306 1307ACCGUAAGUGGC(c3)GCACGGAAUU (guide)

The results of this example are indicated in FIGS. 20A and 20B. A numberof the segmented miRNA mimetics of this example showed increasedknockdown in comparison to the non-segmented duplex miR-124.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

What is claimed is:
 1. A double-stranded oligonucleotide moleculerepresented by Formula III:

wherein (a) the molecule comprises a contiguous passenger strand and aguide strand where each line and its adjacent “N” represent a contiguousstretch of nucleotides, each of “X1,” “X2” and “X3” represent the numberof nucleotides in each stretch, “GIN” represents a discontinuity in theguide strand, “Y1” represents a number of nucleotide positions in thediscontinuity, and each group of dashed lines “

” and its adjacent “(W)” represents a terminal overhang that isoptionally present, and each of “Z1” and “Z2” represents the number ofoverhanging nucleotides; (b) X1 is an integer from 16 to 26, X2 is aninteger from 2 to 21, X3 is an integer from 2 to 9, Y1 is an integerfrom 0 to 6, provided that the sum of X2, X3 and Y1 is an integer from16 to 26; Z1 and Z2 are independently integers from 0 to 4; (c) N_(X2)and N_(X3) together comprise sequence having at least 6 contiguousnucleotides of the seed sequence of a miRNA sequence having any of SEQID NOs: 1-1090; (d) N_(X2) and N_(X3) together comprise sequence havingat least 50% homology to the miRNA sequence; and (e) N_(X1) comprisessequence having at least 50% complementarity to the miRNA sequence. 2.The double stranded oligonucleotide molecule of claim 1, wherein G/Ncomprises a nick in the guide strand.
 3. The double strandedoligonucleotide molecule of claim 1, wherein G/N comprises one or moregap(s) in the guide strand.
 4. The double stranded oligonucleotidemolecule of claim 1, wherein G/N comprises one or more nonnucleotidesubstitutions in the guide strand.
 5. The double strandedoligonucleotide molecule of claim 1, wherein G/N comprises one or morenonnucleotide insertions in the guide strand.
 6. The double strandedoligonucleotide molecule of claim 1, wherein NX2 and NX3 togethercomprise a sequence having at least 7 contiguous nucleotides of the seedsequence of the miRNA.
 7. The double stranded oligonucleotide moleculeof claim 1, wherein NX2 and NX3 together comprise a sequence having atleast 8 contiguous nucleotides of the seed sequence of the miRNA.
 8. Thedouble stranded oligonucleotide molecule of claim 1, wherein NX2 and NX3together comprise a sequence having at least 60, 70, 80, or 90% homologyto the miRNA sequence.
 9. The double stranded oligonucleotide moleculeof claim 1, wherein NX 1 comprises a sequence having at least 60, 70,80, or 90% complementarity to the miRNA sequence.
 10. A compositioncomprising the double stranded oligonucleotide molecule of claim 1 and apharmaceutically acceptable carrier, diluent, excipient, adjuvant,emulsifier, buffer, stabilizer, or preservative.
 11. The double strandedoligonucleotide molecule of claim 5, wherein the one or morenonnucleotide insertions in the guide strand comprise an alkyl moiety.12. The double stranded oligonucleotide molecule of claim 11, whereinthe alkyl moiety comprises any of C₁-C₂₀.
 13. The double strandedoligonucleotide molecule of claim 11, wherein the alkyl moiety comprisesC₃, C₄, C₅, or C₆.
 14. A double-stranded oligonucleotide moleculerepresented by Formula III:

wherein (a) the molecule comprises a contiguous passenger strand and aguide strand where each line and its adjacent “N” represent a contiguousstretch of nucleotides, each of “X1,” “X2” and “X3” represent the numberof nucleotides in each stretch, “GIN” represents a discontinuity in theguide strand, “Y1” represents a number of nucleotide positions in thediscontinuity, and each group of dashed lines “

” and its adjacent “(W)” represents a terminal overhang that isoptionally present, and each of “Z1” and “Z2” represents the number ofoverhanging nucleotides; (b) X₁ is an integer from 16 to 26, X2 is aninteger from 2 to 21, X3 is an integer from 2 to 9, Y1 is 0, 1, or 2,provided that the sum of X2, X3 and Y1 is an integer from 16 to 26; Z1and Z2 are independently integers from 0 to 4; (c) NX2 and NX3 togethercomprise sequence having at least 6 contiguous nucleotides of the seedsequence of a miRNA sequence having any of SEQ ID NOs: 1-1090; (d) NX2and NX3 together comprise sequence having at least 50% homology to themiRNA sequence; (e) NX1 comprises sequence having at least 50%complementarity to the miRNA sequence; and (f) G/N comprises an alkylmoiety.
 15. The double stranded oligonucleotide molecule of claim 14,wherein the alkyl moiety comprises any of C₁-C₂₀.
 16. The doublestranded oligonucleotide molecule of claim 14, wherein the alkyl moietycomprises C₃, C₄, C₅, or C₆.
 17. The double stranded oligonucleotidemolecule of claim 1, wherein NX3 comprises sequence having at least 6contiguous nucleotides of the seed sequence.